Anti-invasive and anti-angiogenic compositions

ABSTRACT

A peptide compound having the sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu [SEQ ID NO:2] or a substitution variant, addition variant or other chemical derivative thereof inhibits cell invasion, endothelial tube formation or angiogenesis in vitro. A number of substitution variants and addition variants of this peptide, preferably capped at the N- and C-termini, as well as peptidomimetic derivates, are useful for treating diseases and conditions mediated by undesired and uncontrolled cell invasion and/or angiogenesis. Pharmaceutical compositions comprising the above peptides and derivatives are administered to subjects in need of such treatment in a dosage sufficient to inhibit invasion and/or angiogenesis. The disclosed compositions and methods are particularly useful for suppressing the growth and metastasis of tumors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/600,302, filed Nov. 15, 2006, now U.S. Pat. No. 7,807,621, which inturn is a continuation of U.S. patent application Ser. No. 10/235,552,filed Sep. 6, 2002, now abandoned, which in turn is a divisionalapplication of U.S. patent application Ser. No. 09/437,136, filed Nov.10, 1999, now U.S. Pat. No. 6,696,416, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 08/900,327,filed Jul. 25, 1997, now U.S. Pat. No. 5,994,309.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention in the fields of biochemistry, organic chemistry andmedicine relates to peptide compounds and methods of their use to treatdiseases and conditions associated with movement, migration and adhesionof cells including diseases that involve angiogenesis such as tumorinvasion and metastasis.

2. Description of the Background Art

Several disease processes have been demonstrated to require the invasionor migration of cells as part of their pathology. These include tumorinvasion, tumor metastasis, pathological angiogenesis, inflammation, andendometriosis (Liotta et al., 1991; Fox et al., 1996; Osborn, 1990;Mareel et al., 1990; Aznavoorian et al., 1993; Lennarz and Strittmatter,1991; Fernandez-Shaw et al., 1995).

In the case of tumor angiogenesis, quiescent endothelial cells canbecome motile in response to a variety of angiogenic growth factors aswell as to changes in the basement membrane induced by tumor cells andvarious accessory cells found within a tumor (Blood and Zetter, 1990;Liotta et al., 1991; Odedra and Weiss, 1991). Neovascularization of atumor enables the metastatic spread of aggressive tumor cells by (1)providing a route of escape for the metastatic cells as well as (2)nurturing the tumor by providing a growth-conducive environment(Cornelius et al., 1995; Blood and Zetter, 1990; Weaver et al., 1997;Weinstat-Saslow and Steeg, 1994; Leek et al., 1994).

The process of tumor metastasis may be viewed as bi-directional,comprising the following steps

-   -   (1) endothelial cells migrate into a tumor in response to a        chemotactic gradient produced by the tumor cells or by accessory        cells (stromal cells, leukocytes); and    -   (2) aggressive tumor cells concomitantly invade toward the        developing neovasculature.

The process of invasion may further fuel angiogenesis by the proteolyticrelease of growth/angiogenic factors bound to extracellular matrix(ECM), including basic fibroblast growth factor (bFGF), vascularendothelial growth factor (VEGF), and hepatocyte growth factor (HGF) aswell as other factors including interleukin-8 (IL-8) andgranulocyte-macrophage colony stimulating factor (GM-CSF). Alsogenerated are proteolytic fragments of the ECM which are themselveschemotactic for both tumor cells and endothelial cells (Fox et al.,1996; Leek et al., 1994; Vlodaysky et al., 1990; Sweeney et al., 1991;Taipale and Keski-Oja, 1997).

It has been suggested that only 1-2% of the total cells in a tumor arecapable of metastasis. As this statement is based on a static view ofthe tumor phenotype, it is probably inaccurate. In reality, metastasisappears to depend on disseminated tumor cells becoming exposed to anenvironment which supports their spread and survival (Weaver et al.,1997). In the majority of patients presenting with a clinicallydetectable primary tumor, metastasis has already occurred (Welch, 1997).Metastatic disease occurs when the disseminated foci of tumor cells seeda tissue which supports their growth and propagation, and this secondaryspread of tumor cells is responsible for the morbidity and mortalityassociated with the majority of cancers. Clinical management ofmetastatic disease is often unsuccessful with conventional cytotoxictherapies. Metastasis differs substantially from the growth of theprimary tumor in that it involves the simultaneous outgrowth of manyfoci which are phenotypically similar from the standpoint of theiraggressiveness. This outgrowth is dependent on the ability of cells thathave metastasized to invade locally and to recruit neovessels.

By preventing interaction of adhesion molecules, the important processof cell mitigation/invasion and angiogenesis can be diminished orhalted, with a number of important consequences for those diseases andconditions which are caused in part by undesirable cell migration,invasion and angiogenesis. In addition to vascular phenomena, such cellmigration/invasion is important in tumor metastasis, which can besuppressed by the compositions and methods disclosed herein.Administration of effective amounts of these compositions will alsodisrupt the molecular interactions required for angiogenesis.

The art recognizes the need for novel treatments of subjects withcancer, in particular patients with metastatic cancer who have thepoorest prognosis. Such treatment should be as devoid as possible ofundesired side effects such as those associated with conventionalchemotherapy and some of the experimental biotherapies. The presentinvention is directed to this objective. Inhibition of tumor cellinvasion and endothelial cell migration (an important component of theangiogenic process) provide a novel approach to treating subjects withmetastatic cancer. By inhibiting the local spread of tumor cells andangiogenesis at metastatic sites, metastatic foci should be induced toregress due to deprivation of their blood supply thus encouraging thesubsequent expression of the cells' endogenous apoptotic program.

Furthermore, the inhibition of invasion of tissue by leukocytes and theconcomitant angiogenesis would be useful for treating inflammation andother disease processes wherein cellular invasiveness is part of thepathogenic process. Inflammation and tumor invasion and metastasis andangiogenesis are known to involve similar mechanisms and extracellularfactors (Liotta et al., 1991; Fox et al., 1996; Osborn, 1990, Mareel etal., 1990; Aznavoorian et al., 1993; Lennarz and Strittmatter, 1993).

Blasi et al. (U.S. Pat. No. 5,416,006) discloses plasminogen activatorsand their chemical modification, in particular phosphorylated uPA andtPA as thrombolytic agents. These workers examined phosphorylated uPA bygenerating tryptic phosphopeptides therefrom and noted the existence ofKPSSPPEELK [SEQ ID NO:1] (corresponding to positions 136-145 of uPA).This decapeptide was not tested for any function, nor ascribed anyproperties of functional relevance. More importantly, as disclosedherein, this peptide (unphosphorylated), capped or uncapped, is inactivein an in vitro assay of cell invasion.

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art. All statements as to the dateor representation as to the contents of these documents is based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for treatingdiseases and processes mediated by undesired and uncontrolled cellinvasion and/or angiogenesis by administering to an animal a compositioncomprising an oligopeptide, chemical derivative or peptidomimetic in adosage sufficient to inhibit the invasion and/or angiogenesis. Thepresent invention is particularly useful for treating or for suppressingthe growth of tumors. Administration of the composition to a human orsubject with prevascularized metastasized tumors will prevent the growthor expansion of those tumors.

Thus, the present invention is directed to a peptide compound having thesequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu (also abbreviated in singleletter amino acid code as KPSSPPEE) [SEQ ID NO:2] or a substitutionvariant, addition variant or other chemical derivative thereof. Thepreferred peptide, variant or derivative is “capped” at the amino andcarboxyl termini, wherein (a) acetyl (abbreviated as “Ac”) is bound tothe N at the amino-terminus and (b) an amido group (abbreviated as “Am”)is bound to the C-terminal carboxyl group. In general, this cappedpeptide will be written “Ac-KPSSPPEE-Am” (SEQ ID NO: 2) throughout thisdocument using the single letter amino acid code and indicating theblocking groups as Ac and Am. This compound is also designated “Å6” andwill therefore be referred to by this name as well.

The peptide, variant or derivative of this invention has one or more ofthe following activities:

-   -   (a) at least about 20% of the biological activity of        Ac-KPSSPPEE-Am (SEQ ID NO: 2) in one or more of the following in        vitro bioassays: (i) invasion in a Matrigel® assay; (ii)        endothelial tube formation on Matrigel®, or (iii) endothelial        tube formation on a fibrin matrix in the presence of basic        fibroblast growth factor and vascular endothelial growth factor;        or    -   (b) binding activity such that it competes with labeled        Ac-KPSSPPEE-Am (SEQ ID NO: 2) for binding to a cell or molecule        which has a binding site for Ac-KPSSPPEE-Am (SEQ ID NO: 2).

In a preferred embodiment, the peptide or peptide variant is capped atboth ends with an N-terminal acetyl group and a C terminal amide group.

A preferred substitution or addition variant of the peptide, or achemical derivative of the variant, has an amino acid sequence selectedfrom the group consisting of:

-   -   (a) SEQ ID NO:2 wherein the Glu at position 7 or 8 or both is        replaced by one or any two of the substituent amino acids Gln,        Asp or Asn;    -   (b) SEQ ID NO:2 wherein Ser at position 3 or 4 or both is        replaced by one or any two of the substituent amino acids Thr,        Ala, Gly, hSer (homoserine) or ValβOH (β-hydroxyvaline);    -   (c) SEQ ID NO:2 wherein the Lys at position 1 is replaced by        His, Arg, Gln, Orn (ornithine), Cit (citrulline) or Hci        (homocitrulline);    -   (d) SEQ ID NO:2 wherein the Pro at position 2, 5 or 6 is        replaced by Hyp (hydroxyproline);    -   (e) an addition variant of SEQ ID NO:2, wherein Leu, Ile, Val,        Nva (norvaline), Nle (norleucine), Met, Ala, or Gly is added to        the C-terminal Glu or to any C-terminal substituent for Glu at        position 8 as disclosed above.    -   (f) an addition variant of SEQ ID NO:2, wherein any of the        following peptides are added to the C-terminal Glu or to the C        terminal substituent for Glu at position 8: Leu-(Gly)_(n)(SEQ ID        NO: 12); Ile-(Gly)_(n) (SEQ ID NO: 13); Val-(Gly)_(n) (SEQ ID        NO: 14); Nva-(Gly)_(n) (SEQ ID NO: 15); or Nle-(Gly)_(n) (SEQ ID        NO: 16), wherein n=1-10.    -   (g) an addition variant of SEQ ID NO:2 wherein one or more of        the following residues or peptides is added to the N-terminal        Lys, or to any N-terminal substituent of Lys at position 1 as        disclosed: Gly, Lys-(Gly). (SEQ ID NO: 17); Tyr-(Gly)_(n) (SEQ        ID NO: 18); or Gly-(Gly)_(n) (SEQ ID NO: 19), wherein n=1-10;        and    -   (h) a combination of one or more of (a)-(g).

In a preferred embodiment, the chemical derivative above is apeptidomimetic agent.

Also provided is a multimer of the peptide or variant above, which, whenthe peptide is not a variant, has the formula:(KPSSPPEE-X_(m))_(n)-KPSSPPEE (SEQ ID NO: 22) wherein X is selected fromthe group consisting of C₁-C₂₀ alkyl, C₁-C₂₀ alkenyl, C₁-C₂₀ alkynyl,C₁-C₂₀ polyether containing up to 9 oxygen atoms and Gly_(m), andwherein m=0 or 1, n=1-100 and z=1-10.

The invention is further directed to a pharmaceutical composition usefulfor inhibiting invasion of tumor cells or angiogenesis, comprising (a)any of the above peptides, variants or chemical derivatives including apeptidomimetic or a multimeric peptide and (b) a pharmaceuticallyacceptable carrier or excipient.

Also included is a method for inhibiting the invasiveness of tumor cellscomprising contacting the cells with an effective amount of a peptide,variant or derivative as above.

In another embodiment, a method is provided for inhibiting tumorinvasion or metastasis in a subject comprising administering to thesubject any of the above pharmaceutical compositions.

Also provided is a method for inhibiting cell migration, invasion,migration-induced cell proliferation or angiogenesis in a subject havinga disease or condition associated with undesired cell migration,invasion, migration-induced proliferation, or angiogenesis comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition as described above

In any of the foregoing methods, the disease or condition being treatedmay be primary tumor growth, tumor invasion or metastasis,atherosclerosis, post-balloon angioplasty vascular restenosis, neointimaformation following vascular trauma, vascular graft restenosis, fibrosisassociated with a chronic inflammatory condition, lung fibrosis,chemotherapy-induced fibrosis, wound healing with scarring and fibrosis,psoriasis, deep venous thrombosis, or another disease or condition inwhich angiogenesis is pathogenic. The treatment methods are mostpreferred for tumor growth, invasion or metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the inhibitory effect ofAc-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Am (SEQ ID NO: 2) (5 μM) on the invitro invasion of both human (PC-3) and rat (Mat BIII) tumor cell linesin a Matrigel® system as described in the Examples. The left bar of eachpair is the control group and the right bar represents cells respondingin the presence of the peptide.

FIG. 2 is a graph showing the effect of various peptides on the in vitroinvasion of PC-3 cells in a Matrigel® system as described in theExamples. All compounds were tested at a 5 μM. The following peptideswere examined: Ac-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Am (Ac-KPSSPPEE-Am)[SEQ ID NO:2] and the following variants of SEQ ID NO:2:Ac-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Am (Ac-KPSSPPE-Am) (SEQ ID NO: 5),Ac-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Am (Ac-PSSPPEE-Am) (SEQ ID NO: 4) as wellas SEQ ID NO:1 (Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Leu-Lys) either capped(Ac-KPSSPPEELK-Am) (SEQ ID NO: 1) or uncapped (KPSSPPEELK) (SEQ ID NO:1).

FIG. 3 shows that Å6 inhibits the activation of plasminogen in a clotlysis assay. Fibrin clots were formed by supplementing plasma with[¹²⁵I] fibrinogen. Clotting was initiated by adding thrombin and calciumto the plasma mixture. Clots were washed to remove any freeradioactivity, plasminogen and the activator to be tested were added tothe clot and the release of [¹²⁵I] fibrin degradation products wasmonitored. The data presented in this figure is at 30 minutes afterinitiation of clot lysis. scuPA-suPAR: dose response with Å6 on clotlysis by scuPA-suPAR complex; scuPA: dose response on clot lysis byscuPA alone; scuPA/suPAR+scrambled pep: dose response with a scrambledversion of Å6 (Ac-PSESPEKP-Am) (SEQ ID NO: 10).

FIGS. 4 and 5 show the results of HUVEC proliferation assays. HUVEC wereplated on gelatin and allowed to adhere for 4 hrs in the presence of 2%FBS. bFGF (1 ng/mL) and the test compounds were added after 4 hours andthe % proliferation (where bFGF induced proliferation was 100% andproliferation in the absence of bFGF was 0) was determined using MTS.FIG. 4: the Å6 (50 μg/mL) concentration was kept constant and theconcentration of cisplatin (“CDDP”) was varied; FIG. 5: CDDP was keptconstant at a sub-optimal dose (1 μg/mL) and Å6 was varied.

FIG. 6 shows the migration of HMVEC on Type I collagen. Membranescontained in transwell chambers (8.0 μm) were coated with Type ICollagen. Microvessel endothelial cells (HMVECs, 2×10⁵/well) were addedto the top chamber of each well and bFGF (10 ng/mL) was added to eachbottom chamber as a chemoattractant. Inhibitor was added to bothchambers and the migration allowed to proceed for 6 hrs at 37° C. Thetop of each filter was scraped to remove cells that had not migrated andthe cells adhering to the bottom of the filter were fixed and stainedusing DiffQuick and the cells were quantitated by counting. The resultsare expressed as a percentage of the cells migrated in response to bFGFalone.

FIG. 7 shows that Å6 inhibits the invasion of Mat B-III rat breastcancer cells and MDA-MB-231 human breast cancer cells through Matrigel.

FIG. 8 shows the inhibition of Mat B-III invasion by Å6 and itsvariants. A: Control (no compound); B: Å6; C: Å6 lacking the C-terminalGlu; D: Å6 lacking the N-terminal Lys; E: Å6 extended by LeuLys at theC-terminus (corresponds to amino acids 144 and 145 in uPA); F: same as Eexcept C-terminal is not capped; G: Å6, N and C termini uncapped; H: Å6extended by Leu at C-terminus (capped).

FIG. 9 is a PAGE gel pattern of biotin-Å6 crosslinked to MDA-MB-231cells. Cells were incubated in the presence of biotin-Å6 (200 μM) for 2hrs. DSS (1 mM) was added and cross-linking was allowed to proceed for15 minutes. The cells were washed and extracted using PBS/1% TritonX-114. Cell particulate was removed by centrifugation and thesupernatants were heated to 37° C. for 5 minutes to induce phaseseparation. Aqueous and detergent (membrane) phases were separated bycentrifugation and each phase was resolved by SDS-PAGE and analyzed bywestern blot using strepatavidin-HRPO.

A: Aqueous phase, 2^(nd) wash; B: Detergent Phase; C: Aqueous phase,1^(st) wash; D: MW markers; E: Biotin-uPA.

FIG. 10 shows that Å6 inhibits angiogenesis in a CAM assay. Filter diskssaturated with either compound or compound+bFGF (0.3 ng) were placed onthe CAM (7 day old) and vessel formation was observed for 4 days. Majorvessels were quantitated at this time.

FIG. 11 shows the results of CAM assays. A: Control CAM; B: CAM treatedwith 1.2 μg/disk of Å6.

FIGS. 12 and 13 show that Å6 inhibits Mat B-III tumor growth andmetastasis. FIG. 12: The volume of the primary challenge tumor wasdetermined using caliper measurements. Inhibition of tumor growth by Å6was most effective when the tumors were small. FIG. 13: Macroscopicmetastasis was also inhibited by Å6 treatment. Control animals receivedvehicle (PBS) only.

FIG. 14 shows results of in vitro and in vivo studies using Å6 and TAM.TopPanel: Invasion of Mat B-III cells through Matrigel A: Control; B:TAM (1 μM); C: Å6 (5 μM); D: Å6 (50 μM); E: Å6 (50 μM)+TAM (1 μM).Bottom Panel: Mat B-III tumor bearing animals were treated with TAM (3mg/kg/day), Å6 (75 mg/kg/day) or a combination of TAM+Å6, Tumor volumeswere determined using caliper measurements. Control received vehicle(PBS) only.

FIG. 15 shows that Å6 inhibits the growth of primary challenge tumors inan MDA-MB-231 xenograft model. Nude mice (n=5 per group) were challengedwith 2×10⁵ tumor cells co-injected into the mammary fat pad of the micewith Matrigel. Treatment of the mice with Å6 (IP, 1 mg bid) wasinitiated when the tumor nodules had become palpable (beginning of week1 on graph). Control animals received vehicle (PBS) only.

FIG. 16 shows results of combination therapy of U87 tumors inoculateds.c. in nude mice. U87 cells (1×10⁵) were inoculated sc on the back of anude mouse (n=5). Tumors were staged to 50-100 mm³ (day 0) at which timetreatment was started with either Å6 alone (75 mg/kg/day given IP bid),cisplatin (CDDP) alone (3 mg/kg/day given every other day from day 4×6administrations), or a combination of Å6+CDDP. Tumor volumes weredetermined using caliper measurements. Treatment was discontinued at day20 and the mice were euthanized one week later.

FIG. 17 shows the number of tumor foci using Ki-67 staining of U87tumors. Formalin-fixed sections were stained with mouse anti-Ki-67followed by peroxidase detection. Positive foci were quantitated bycapturing digital images of the slides and determining the number ofpixels associated with the staining on each slide.

FIG. 18 shows the effects of Å6 on orthotopically inoculated human U87GBM cells. U87 cells were inoculated into the ventricles of nude miceand treatment was initiated 72 hrs after inoculation. Tumor volume wasdetermined using caliper measurements after euthanasia and necropsy.

FIG. 19 shows the dose dependent response of U87 tumor growth to Å6.Mice (n=4 per group) were treated as previously described and treatmentwas discontinued after 21 days.

FIG. 20 shows the effect of Å6 and CDDP on survival of mice withorthotopically inoculated U87 GBM tumors. A: Control; B: Å6 only; C:CDDP only; D: Å6+CDDP.

FIG. 21 shows the results of combination treatment of 3LL tumors withÅ6+cyclophosphamide. 3LL cells (1.5×10⁵ cells) were injected into thetail vein of C57/B1 mice. Treatment with Å6 (75 mg/kg/day IP b.i.d.) andcyclophosphamide (4 mg/kg IP on day 4) was continued for 19 days.Animals were euthanized on day 20 and the lungs removed and analyzedmacroscopically and histologically for the presence of tumor. Theaverage number of tumors in each group (n=7) is shown.

FIG. 22 shows the plasma concentration of Å6 in mice following a singleintravenous injection of 37.5 mg/kg.

FIG. 23 shows the plasma concentration of Å6 in Cynomolgus monkeysfollowing a single intravenous injection of 37.5 mg/kg.

FIG. 24 shows allometric scaling of plasma clearance in animals.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have discovered a novel peptide and relatedcompounds which act as inhibitors of angiogenesis and invasiveness andhave devised various methods for using this peptide for diagnosis,therapy and receptor identification. The peptide is a potent andspecific inhibitor of (a) cell invasion, (b) angiogenesis at tumor sitesincluding sites of metastasis, and (c) inflammatory responses.

In addition, the peptide and its derivatives are designed to be highlysoluble in aqueous buffer and body fluids but not in lipids. Thisproperty limits non-specific partitioning into membranes. Non-specificpartitioning of compounds into and across membranes is a frequent causeof toxicity. The compounds of this invention have minimal toxicitybecause, owing to their possessing Coulombic charge, they are notexpected to partition into cells. The target(s) of the compositions areextracellular, and method(s) of this invention are predicated on thecompositions acting first in the extracellular space. Therefore, it isdesirable to maintain the compounds in the extracellular space.

Additional pharmacological advantage is obtained due to the highsolubility limit of the compounds, allowing their delivery in highconcentrations in the absence of co-solvents or extraordinaryexcipients.

Compounds of the invention have been shown by the present inventors (seeExample II) to block the invasion of both human and rat tumor cells invitro in the Matrigel® system.

In addition, they block endothelial cell tube formation in response tobFGF and VEGF in either a fibrin matrix or when the endothelial cellsare plated on Matrigel®.

The compounds of the invention also inhibit experimental metastasis in axenograft model in nu/nu mice using the human prostatic carcinoma cellline, PC-3, transfected with the green fluorescent protein (GFP) as areporter. Finally, the compounds also inhibit tumor progression,spontaneous metastasis and angiogenesis in a syngeneic rat model ofbreast cancer.

The Peptide Compositions

The original inhibitory capped peptide discovered by the presentinventors has 8 amino acid residues with a molecular weight of 911 Da.This preferred peptide is characterized by the sequence:

[SEQ ID NO: 2] CH₃CO-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-NH₂

The amino and carboxyl termini are preferably blocked or “capped” withacetyl (CH₃CO—, bound to the amino-terminal N; also abbreviated as “Ac”)and amido (—NH₂ bound to the C-terminal carboxyl group; also abbreviatedas “Am”), respectively. This peptide will also be referred to below insingle letter code indicating the blocking groups as Ac and Am groups:Ac-KPSSPPEE-Am (SEQ ID NO: 2).

The N-terminal capping function is preferably in a linkage to theterminal amino group and may be selected from the group consisting of:

formyl;

alkanoyl, having from 1 to 10 carbon atoms, such as acetyl, propionyl,butyryl;

alkenoyl, having from 1 to 10 carbon atoms, such as hex-3-enoyl;

alkynoyl, having from 1 to 10 carbon atoms, such as hex-5-ynoyl;

aroyl, such as berizoyl or 1-naphthoyl;

heteroaroyl, such as 3-pyrroyl or 4-quinoloyl;

alkylsulfonyl, such as methanesulfonyl;

arylsulfonyl, such as benzenesulfonyl or sulfanilyl;

heteroarylsulfonyl, such as pyridine-4-sulfonyl;

substituted alkanoyl, having from 1 to 10 carbon atoms, such as4-aminobutyryl;

substituted alkenoyl, having from 1 to 10 carbon atoms, such as6-hydroxy-hex-3-enoyl;

substituted alkynoyl, having from 1 to 10 carbon atoms, such as3-hydroxy-hex-5-ynoyl;

substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-naphth-2-oyl;

substituted heteroaroyl, such as2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazolin-6-oyl;

substituted alkylsulfonyl, such as 2-aminoethanesulfonyl;

substituted arylsulfonyl, such as 5-dimethylamino-1-naphthalenesulfonyl;

substituted heteroarylsulfonyl, such as1-methoxy-6-isoquinolinesulfonyl;

carbamoyl or thiocarbamoyl;

substituted carbamoyl (R′—NH—CO) or substituted thiocarbamoyl (R′—NH—CS)

wherein R′ is alkyl, alkenyl, alkynyl, aryl, heteroaryl, substitutedalkyl, substituted alkenyl, substituted alkynyl, substituted aryl, orsubstituted heteroaryl;

substituted carbamoyl (R′—NH—CO) and substituted thiocarbamoyl(R′—NH—CS) wherein R′ is alkanoyl, alkenoyl, alkynoyl, aroyl,heteroaroyl, substituted alkanoyl, substituted alkenoyl, substitutedalkynoyl, substituted aroyl, or substituted heteroaroyl, all as abovedefined;

Lys-(Gly)_(n) where n=1-4 (SEQ ID NO: 20); or Tyr-(Gly)_(n) where n=1-4(SEQ ID NO: 21).

The C-terminal capping function can either be in an amide bond with theterminal carboxyl or in an ester bond with the terminal carboxyl.Capping functions that provide for an amide bond are designated as NR¹R²wherein R¹ and R² may be independently drawn from the following group:

hydrogen;

alkyl, preferably having from 1 to 10 carbon atoms, such as methyl,ethyl, isopropyl;

alkenyl, preferably having from 1 to 10 carbon atoms, such asprop-2-enyl;

alkynyl, preferably having from 1 to 10 carbon atoms, such asprop-2-ynyl;

substituted alkyl having from 1 to 10 carbon atoms, such ashydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, halogenoalkyl,cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,alkanoylalkyl, carboxyalkyl, carbamoylalkyl;

substituted alkenyl having from 1 to 10 carbon atoms, such ashydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl,halogenoalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl,dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl;

substituted alkynyl having from 1 to 10 carbon atoms, such ascyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl,alkanoylalkynyl, carboxyalkynyl, carbamoylalkynyl;

aroylalkyl having up to 10 carbon atoms, such as phenacyl or2-benzoylethyl

aryl, such as phenyl or 1-naphthyl;

heteroaryl, such as 4-quinolyl;

alkanoyl having from 1 to 10 carbon atoms, such as acetyl or butyryl;

aroyl, such as benzoyl;

heteroaroyl, such as 3-quinoloyl;

OR′ or NR′R″ where R′ and R″ are independently hydrogen, alkyl, aryl,heteroaryl, acyl, aroyl, sulfonyl, sulfinyl, or SO₂—R″ or SO—R″ where R″is substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, oralkynyl.

Capping functions that provide for an ester bond are designated as OR,wherein R may be: alkoxy; aryloxy; heteroaryloxy; aralkyloxy;heteroaralkyloxy; substituted alkoxy; substituted aryloxy; substitutedheteroaryloxy; substituted aralkyloxy; or substituted heteroaralkyloxy.

Either the N-terminal or the C-terminal capping function, or both, maybe of such structure that the capped molecule functions as a prodrug (apharmacologically inactive derivative of the parent drug molecule) thatundergoes spontaneous or enzymatic transformation within the body inorder to release the active drug and that has improved deliveryproperties over the parent drug molecule (Bundgaard, 1985).

Judicious choice of capping groups allows the addition of otheractivities on the peptide. For example, the presence of a sulfhydrylgroup linked to the N- or C-terminal cap will permit conjugation of thederivatized peptide to other molecules.

Capping of the peptide is intended primarily to increase plasma halflife, as has been demonstrated for many peptides (e.g., Powell et al.,Ann Repts Med. Chem. 28:285-294, 1993). Any capping group which servesthis function is intended. However, the uncapped form is still useful asa template for peptidomimetic design (see below) and may have acceptableactivity in vitro.

Production of Peptides and Derivatives

General Chemical Synthetic Procedures

The peptides of the invention may be prepared using recombinant DNAtechnology. However, given their length, they are preferably preparedusing solid-phase synthesis, such as that generally described byMerrifield, J. Amer. Chem. Soc., 85:2149-54 (1963), although otherequivalent chemical syntheses known in the art are also useful.Solid-phase peptide synthesis may be initiated from the C-terminus ofthe peptide by coupling a protected α-amino acid to a suitable resin.Such a starting material can be prepared by attaching anα-amino-protected amino acid by an ester linkage to a chlormethylatedresin or to a hydroxymethyl resin, or by an amide bond to a BHA resin orMBHA resin.

The preparation of the hydroxymethyl resin is described by Bodansky etal., 1966. Chlromethylated resins are commercially available from BioRadLaboratories, Richmond, Calif. and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart et al., 1969. BHAand MBHA resin supports are commercially available and are generallyused only when the desired polypeptide being synthesized has anunsubstituted amide at the C-terminus.

The amino acids can be coupled to the growing peptide chain usingtechniques well known in the art for the formation of peptide bonds. Forexample, one method involves converting the amino acid to a derivativethat will render the carboxyl group of the amino acid more susceptibleto reaction with the free N-terminal amino group of the growing peptidechain. Specifically, the C-terminal of the protected amino acid can beconverted to a mixed anhydride by the reaction of the C-terminal withethyl chloroformate, phenyl chloroformate, sec-butyl chloroformate,isobutyl chloroformate, or pivaloyl chloride or the like chlorides.Alternatively, the C-terminal of the amino chlorides. Alternatively, theC-terminal of the amino acid can be converted to an active ester, suchas a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, apentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimideester, or an ester formed from 1-hydroxybenzotriazole. Another couplingmethod involves the use of a suitable coupling agent, such asN,N′-dicyclohexylcarbodiimide or N,N′-diisopropylcarbodiimide. Otherappropriate coupling agents, apparent to those skilled in the art, aredisclosed in Gross et al. 1979, which is hereby incorporated byreference.

The α-amino group of each amino acid employed in the peptide synthesismust be protected during the coupling reaction to prevent side reactionsinvolving their active α-amino function. Certain amino acids containreactive side-chain functional groups (e.g., sulfhydryl, amino,carboxyl, and hydroxyl) and such functional groups must also beprotected with suitable protecting groups to prevent a chemical reactionfrom occurring at either (1) the α-amino group site or (2) a reactiveside chain site during both the initial and subsequent coupling steps.

In the selection of a particular protecting group to be used insynthesizing the peptides, the following general rules are typicallyfollowed. Specifically, an α-amino protecting group (1) should renderthe α-amino function inert under the conditions employed in the couplingreaction, (2) should be readily removable after the coupling reactionunder conditions that will not remove side-chain protecting groups andwill not alter the structure of the peptide fragment, and (3) shouldsubstantially reduce the possibility of racemization upon activation,immediately prior to coupling.

On the other hand, a side-chain protecting group (1) should render theside chain functional group inert under the conditions employed in thecoupling reaction, (2) should be stable under the conditions employed inremoving the α-amino protecting group, and (3) should be readilyremovable from the desired fully-assembled peptide under reactionconditions that will not alter the structure of the peptide chain.

It will be apparent to those skilled in the art that the protectinggroups known to be useful for peptide synthesis vary in reactivity withthe agents employed for their removal. For example, certain protectinggroups, such as triphenylmethyl and 2-(p-biphenyl)isopropyloxycarbonyl,are very labile and can be cleaved under mild acid conditions. Otherprotecting groups, such as t-butyloxycarbonyl (BOC), t-amyloxycarbonyl,adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, are less labileand require moderately strong acids for their removal, such astrifluoroacetic, hydrochloric, or boron trifluoride in acetic acid.Still other protecting groups, such as benzyloxycarbonyl (CBZ or Z),halobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl cycloalkyloxycarbortyl,and isopropyloxycarbonyl, are even less labile and require even strongeracids, such as hydrogen fluoride, hydrogen bromide, or borontrifluoroacetate in trifluoroacetic acid, for their removal. Suitableprotecting groups, known in the art are described in Gross et al. 1981.

Among the classes of α-amino acid protecting groups useful forprotecting the α-amino group or for protecting a side chain group areincluded the following.

(1) For an α-amino group, three typical classes of protecting groupsare:

-   -   (a) aromatic urethane-type protecting groups, such as        fluorenylmethyloxycarhonyl (FMOC), CBZ, and substituted CBZ,        such as, p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,        p-bromobenzyloxycarhonyl, and p-methoxybenzyloxycarbonyl,        o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,        2,6-dichlorobenzyloxycarbonyl, and the like;    -   (b) aliphatic urethane-type protecting groups, such as BOC,        t-amyloxycarbonyl, isopropyloxycarbonyl,        2-(p-biphenyl)isopropyloxycarbonyl, allyloxycarbonyl and the        like; and    -   (c) cycloalkyl urethane-type protecting groups, such as        cyclopentyloxycarbonyl, adamantyloxycarbonyl, and        cyclohexyloxycarbonyl.

The preferred α-amino protecting groups are BOC and FMOC.

(2) For the side chain amino group present in Lys, protection may be byany of the groups mentioned above in (1) such as BOC,2-chlorobcenzyloxycarbonyl and the like.

(3) For the guanidino group of Arg, protection may be provided by nitro,tosyl, CBZ, adamantyloxycarbonyl,2,2,5,7,8-pentamethylchroman-6-sulfonyl,2,3,6-trimethyl-4-methoxyphenylsulfonyl, or BOC groups.

(4) For the hydroxyl group of Ser or Thr, protection may be, forexample, by t-butyl; benzyl (BZL); or substituted BZL, such asp-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and2,6-dichlorobenzyl.

(5) For the carboxyl group of Asp or Glu, protection may be, forexample, by esterification using such groups as BZL, t-butyl,cyclohexyl, cyclopentyl, and the like.

(6) For the imidazole nitrogen of His, the benzyloxymethyl (BOM) ortosyl moiety is suitably employed as a protecting group.

(7) For the phenolic hydroxyl group of Tyr, a protecting group such astetrahydropyranyl, tert-butyl, trityl, BZL, chlorobenzyl, 4-bromobenzyl,and 2,6-dichlorobenzyl are suitably employed. The preferred protectinggroup is bromobenzyloxycarhonyl.

(8) For the side chain amino group of Asn or Gln, xanthyl (Xan) ispreferably employed.

(9) For Met, the amino acid is preferably left unprotected.

(10) For the thio group of Cys, p-methoxybenzyl is typically employed.

The first C-terminal amino acid of the growing peptide chain, e.g., Glu,is typically protected at the α-amino position by an appropriatelyselected protecting group such as BOC. The BOC-Glu-(γ-cyclohexyl)-OH canbe first coupled to a benzylhydrylamine resin usingisopropylcarbodiimide at about 25° C. for two hours with stirring or toa chloromethylated resin according to the procedure set forth in Horikiet al., 1978. Following the coupling of the BOC-protected amino acid tothe resin support, the α-amino protecting group is usually removed,typically by using trifluoroacetic acid (TFA) in methylene chloride orTFA alone. The α-amino group de-protection reaction can occur over awide range of temperatures, but is usually carried out at a temperaturebetween about 0° C. and room temperature.

Other standard α-amino group de-protecting reagents, such as HCl indioxane, and conditions for the removal of specific α-amino protectinggroups are within the skill of those working in the art, such as thosedescribed in Lübke et al., 1975, which is hereby incorporated byreference. Following the removal of the α-amino protecting group, theunprotected α-amino group, generally still side-chain protected, can becoupled in a stepwise manner in the intended sequence.

An alternative to the stepwise approach is the fragment condensationmethod in which pre-formed peptides of short length, each representingpart of the desired sequence, are coupled to a growing chain of aminoacids bound to a solid phase support. For this stepwise approach, aparticularly suitable coupling reagent is N,N′-dicyclohexylcarbodiimideor diisopropylcarbodiimide. Also, for the fragment approach, theselection of the coupling reagent, as well as the choice of thefragmentation pattern needed to couple fragments of the desired natureand size are important for success and are known to those skilled in theart.

Each protected amino acid or amino acid sequence is usually introducedinto the solid-phase reactor in amounts in excess of stoichiometricquantities, and the coupling is suitably carried out in an organicsolvent, such as dimethylformamide (DMF), CH₂Cl₂ or mixtures thereof. Ifincomplete coupling occurs, the coupling procedure is customarilyrepeated before removal of the N-amino protecting group in preparationfor coupling to the next amino acid. Following the removal of theα-amino protecting group, the remaining α-amino and side-chain-protectedamino acids can be coupled in a stepwise manner in the intendedsequence. The success of the coupling reaction at each stage of thesynthesis may be monitored. A preferred method of monitoring thesynthesis is by the ninhydrin reaction, as described by Kaiser et al.,1970. The coupling reactions can also be performed automatically usingwell-known commercial methods and devices, for example, a Beckman 990Peptide Synthesizer.

Upon completion of the desired peptide sequence, the protected peptidemust be cleaved from the resin support, and all protecting groups mustbe removed. The cleavage reaction and removal of the protecting group issuitably accomplished concomitantly or consecutively with de-protectionreactions. When the bond anchoring the peptide to the resin is an esterbond, it can be cleaved by any reagent that is capable of breaking anester linkage and of penetrating the resin matrix. One especially usefulmethod is by treatment with liquid anhydrous hydrogen fluoride. Thisreagent will usually not only cleave the peptide from the resin, butwill also remove all acid-labile protecting groups and, thus, willdirectly provide the fully de-protected peptide. When additionalprotecting groups that are not acid-labile are present, additionalde-protection steps must be carried out. These steps can be performedeither before or after the hydrogen fluoride treatment described above,according to specific needs and circumstances.

When a chloromethylated resin is used, the hydrogen fluoridecleavage/de-protection treatment generally results in the formation ofthe free peptide acids. When a benzhydrylamine resin is used, thehydrogen fluoride treatment generally results in the free peptideamides. Reaction with hydrogen fluoride in the presence of anisole anddimethylsulfide at 0° C. for one hour will typically remove theside-chain protecting groups and, concomitantly, release the peptidefrom the resin.

When it is desired to cleave the peptide without removing protectinggroups, the protected peptide-resin can be subjected to methanolysis,thus yielding a protected peptide in which the C-terminal carboxyl groupis methylated. This methyl ester can be subsequently hydrolyzed undermild alkaline conditions to give the free C-terminal carboxyl group. Theprotecting groups on the peptide chain can then be removed by treatmentwith a strong acid, such as liquid hydrogen fluoride. A particularlyuseful technique for methanolysis is that of Moore et al., 1977, inwhich the protected peptide-resin is treated with methanol and potassiumcyanide in the presence of a crown ether.

Other methods for cleaving a protected peptide from the resin when achloromethylated resin is employed include (1) ammoniolysis and (2)hydrazinolysis. If desired, the resulting C-terminal amide or hydrazidecan be hydrolyzed to the free C-terminal carboxyl moiety, and theprotecting groups can be removed conventionally. The protecting grouppresent on the N-terminal α-amino group may be removed either before, orafter, the protected peptide is cleaved from the support. Purificationof the peptides of the invention is typically achieved usingchromatographic techniques, such as preparative HPLC (including reversephase HPLC), gel permeation, ion exchange, partition chromatography,affinity chromatography (including monoclonal antibody columns), and thelike, or other conventional techniques such as countercurrentdistribution or the like.

Amino Acid Substitution and Addition Variants

Also included in this invention are peptides in which at least one aminoacid residue and preferably, only one, has been removed and a differentresidue inserted in its place. For a detailed description of proteinchemistry and structure, see Schulz, G. E. et al., Principles of ProteinStructure, Springer-Verlag, New York, 1979, and Creighton, T. E.,Proteins: Structure and Molecular Principles, W.H. Freeman & Co., SanFrancisco, 1984, which are hereby incorporated by reference. The typesof substitutions which may be made in the peptide molecule of thepresent invention are conservative substitutions and are defined hereinas exchanges within one of the following groups:

1. Small aliphatic, nonpolar or slightly polar residues: e.g., Ala, Ser,Thr, Gly;

2. Polar, negatively charged residues and their amides: e.g., Asp, Asn,Glu, Gln;

3. Polar, positively charged residues: e.g., His, Arg, Lys;

Pro, because of its unusual geometry, tightly constrains the chain.Substantial changes in functional properties are made by selectingsubstitutions that are less conservative, such as between, rather thanwithin, the above groups (or two other amino acid groups not shownabove), which will differ more significantly in their effect onmaintaining (a) the structure of the peptide backbone in the area of thesubstitution (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. Most substitutionsaccording to the present invention are those which do not produceradical changes in the characteristics of the peptide molecule. Evenwhen it is difficult to predict the exact effect of a substitution inadvance of doing so, one skilled in the art will appreciate that theeffect can be evaluated by routine screening assays, preferably thebiological assays described below. Modifications of peptide propertiesincluding redox or thermal stability, hydrophobicity, susceptibility toproteolytic degradation or the tendency to aggregate with carriers orinto multimers are assayed by methods well known to the ordinarilyskilled artisan.

One group of preferred substitution variants of KPSSPPEE (SEQ ID NO: 2)have the Glu at position 7 or 8 (or both) of SEQ ID NO:2 replaced by oneor any two of Gln, Asp or Asn.

Other derivatives may further include substitution of the Ser atposition 3 or 4 (or both) of SEQ ID NO:2 with one or any two of thefollowing: Thr, Ala, Gly, hSer or ValβOH.

Furthermore, the Lys at position 1 of SEQ ID NO:2 may be replaced by Hs,Arg, Gln, Orn, Cit or Hci

Other derivatives have Pro at position 2, 5 or 6 replaced by Hyp(hydroxyproline).

It is noteworthy that any and all combinations of the foregoingsubstitutions are within the scope of this invention.

Also included in this invention are addition variants wherein two ormore residues are added to the C-terminus after Glu (or after any of itsabove substituents) in SEQ ID NO:2. These residues may be Leu-(Gly)_(n)(SEQ ID NO: 12), Ile-(Gly)_(n) (SEQ ID NO: 13), Val-(Gly)_(n), (SEQ IDNO: 14), Nva-(Gly)_(n) (SEQ ID NO: 15), or Nle-(Gly)_(n) (SEQ ID NO:16), wherein Nva is norvaline, Nle is norleucine, and n=1-10.

Also included in this invention are addition variants wherein one ormore residues is/are added to the N-terminus before Lys (or any of itsabove substituents) in SEQ ID NO:2. These residues may be Gly,Lys-(Gly)_(n) (SEQ ID NO: 17), Tyr-(Gly)_(n) (SEQ ID NO: 18), orGly-(Gly)_(n) (SEQ ID NO: 19) wherein n=1-10.

Another preferred derivative of this invention is a 9-mer additionvariant wherein any one of the following amino acids is added to theC-terminus after Glu (or any of its above substituents) in SEQ ID NO:2:Leu, Ile, Val, Nva, Nle, Met, Ala, or Gly.

In general, preferred peptide addition variants may have up to about 30additional amino acids, more preferably about 20, most preferably 11.The functional limitations placed on the peptide variant, and the easeby which these activities can be tested using conventional means, wouldpermit one skilled in the art to ascertain whether an addition (or anyother type of) variant would affect the peptide's activity. In view ofthe structural description provided herein, it would be to determinewhether a peptide variant falls within the scope of this invention.

Uncapped peptides of any of the foregoing sequences having free N- andC-termini, for example, uncapped NH₂-KPSSPPEE-OH [SEQ ID NO:2].

Chemical Derivatives

“Chemical derivatives” of KPSSPPEE [SEQ ID NO:2] contain additionalchemical moieties not normally a part of the peptide. Covalentmodifications of the peptide are included within the scope of thisinvention. Such modifications may be introduced into the molecule byreacting targeted amino acid residues of the peptide with an organicderivatizing agent that is capable of reacting with selected side chainsor terminal residues.

The capped peptides discussed above are examples of preferred chemicalderivatives of the “natural” uncapped peptide. Any of the abovecombination of substitution or addition variants may be capped with anyof the capping groups disclosed herein.

Other examples of chemical derivatives of the peptide follow.

Lysinyl and amino terminal residues are derivatized with succinic orother carboxylic acid anhydrides. Derivatization with a cycliccarboxylic anhydride has the effect of reversing the charge of thelysinyl residues. Other suitable reagents for derivatizingα-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Carboxyl side groups, aspartyl or glutamyl, may be selectively modifiedby reaction with carbodiimides (R—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonia.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the amino group of lysine (Creighton, supra, pp. 79-86),acetylation of the N-terminal amine, and amidation of the C-terminalcarboxyl groups.

For every single peptide sequence disclosed herein, this inventionincludes the corresponding retro-inverso sequence wherein the directionof the peptide chain has been inverted and wherein all the amino acidsbelong to the D-series. For example the retro-inverso analogue of thenatural L-series peptide KPSSPPEE (SEQ ID NO: 2) is EEPPSSPK (SEQ ID NO:11) which is composed of D-series amino acids and in which E is theN-terminus and K is the C-terminus. For example the retro-inversoanalogue of the natural L-series capped peptide Ac-KPSSPPEE-Am (SEQ IDNO: 2) is Ac-EEPPSSPK-Am (SEQ ID NO: 11) which is composed of D-seriesamino acids and in which the N-terminal E is acetylated and theC-terminal K is amidated. The complete range of N-terminal cappinggroups and the complete range C-terminal capping groups specified forthe L-series peptides are also intended for the D-series peptides.

Also included are peptides wherein one or more D-amino acids has/havebeen substituted for one or more L-amino acids. Additionally, modifiedamino acids or chemical derivatives of amino acids may be provided suchthat the peptide contains additional chemical moieties or modified aminoacids not normally a part of a natural protein. Such derivatizedmoieties may improve the solubility, absorption, biological half life,and the like. Moieties capable of mediating such effects are disclosed,for example, in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980).

Multimeric Peptides

The present invention also includes longer peptides in which the basicpeptidic sequence of about 7-9 amino acids is repeated from about two toabout 100 times, with or without intervening spacers or linkers. Amultimer of the peptide KPSSPPEE (SEQ ID NO: 2) is shown by thefollowing formula (KPSSPPEE-X_(m))_(n)-KPSSPPEE (SEQ ID NO: 22) whereinm=0 or 1, n=1-100. X is a spacer group, preferably C₁-C₂₀ alkyl, C₁-C₂₀alkenyl, C₁-C₂₀ alkynyl, C₁-C₂₀ polyether containing up to 9 oxygenatoms or Gly_(z). (z=1-10).

It is understood that such multimers may be built from any of thepeptide variants described herein. Moreover, a peptide multimer maycomprise different combinations of peptide monomers, both KPSSPPEE (SEQID NO: 2) and the disclosed variants thereof. Such oligomeric ormultimeric peptides can be made by chemical synthesis or by recombinantDNA as discussed herein. When produced chemically, the oligomerspreferably have from 2-8 repeats of the basic peptide sequence. Whenproduced recombinantly, the multimers may have as many repeats as theexpression system permits, for example from two to about 100 repeats.

Peptidomimetics

A preferred type of chemical derivative of the peptides described hereinis a peptidomimetic compound which mimics the biological effect ofKPSSPPEE (SEQ ID NO: 2), capped or uncapped. A peptidomimetic agent maybe an unnatural peptide or a non-peptide agent which has thestereochemical properties of KPSSPPEE (SEQ ID NO: 2), capped oruncapped, such that it has the binding activity or biological activityof KPSSPPEE (SEQ ID NO: 2), capped or uncapped. Hence, this inventionincludes compounds wherein a peptidomimetic compound is coupled to apeptide, for example,

X-PPEE (SEQ ID NO: 3)

wherein X is a peptidomimetic which mimics KPSS (SEQ ID NO: 9); thepeptide portion may include a normal or a retro-inverso sequence.

Peptidomimetic compounds, either agonists, substrates or inhibitors,have been described for a number of bioactive peptides such as opioidpeptides, VIP, thrombin, HIV protease, etc. Methods for designing andpreparing peptidomimetic compounds are known in the art (Kempf D J,Methods Enzymol 241:334-354 (1994); Hruby, V. J., Biopolymers 33:1073-82(1993); Wiley, R. A. et al., Med. Res. Rev. 13:327-384 (1993); Claeson,G., Blood Coagul. Fibrinolysis 5:411-436 (1994), which references areincorporated by reference in their entirety). These methods are used toprepare capped or uncapped KPSSPPEE (SEQ ID NO: 2) peptidomimetics whichpossess at least the binding capacity and specificity of the peptide andpreferably also possess the biological activity. Knowledge of peptidechemistry and general organic chemistry available to those skilled inthe art are sufficient for the design and testing of such compounds.

For example, such peptidomimetics may be identified by inspection of thecystallographically-derived three-dimensional structure of a peptide ofthe invention, for example KPSSPPEE (SEQ ID NO: 2), capped or uncapped,either free or bound in complex with its receptor(s). Alternatively, thestructure of a peptide of the invention bound to its receptor(s) can begained by the techniques of nuclear magnetic resonance spectroscopy. Thebetter knowledge of the stereochemistry of the interaction of, say,KPSSPPEE (SEQ ID NO: 2), capped or uncapped, with its receptor willpermit the rational design of such peptidomimetic agents.

All the foregoing peptides, variants and chemical derivatives includingpeptidomimetics and multimeric peptides must have the biologicalactivity and/or the binding activity of KPSSPPEE (SEQ ID NO: 2) asfollows: at least about 20% of the activity of Ac-KPSSPPE-Am (SEQ ID NO:5) in an in vitro assay of cell invasiveness or an in vitro assay ofendothelial tube formation and/or angiogenesis. These activities arecharacterized in greater detail below. Alternatively, or in addition,the peptide, variant or chemical derivatives should compete with labeledAc-KPSSPPEE-Am (SEQ ID NO: 2) for binding to a ligand or binding partnerfor Ac-KPSSPPEE-Am (SEQ ID NO: 2), whether this be a cellular receptor(tested in a binding assay with whole cells or fractions thereof), anisolated receptor or any other Ac-KPSSPPEE-Am-binding molecule (SEQ IDNO: 2).

Moreover, the peptides, variants or derivatives of the present inventiondo not have biological activities previously associated with urokinaseplasminogen activator (uPA). That is they do no block the binding of uPAto the uPA receptor. These peptides lack thrombolytic activity, ahallmark of uPA.

Additional Discussion of Peptides, Variants and Peptidomimetics

Minor modifications of the amino acid sequence might affect activity ifthose modifications are selected purely at random. However, one skilledin the art of peptide and peptidomimetic design would follow awell-established set of “rules” in creating useful variants andderivatives. For example, it is expected that the KPSSPPEE peptidemodified by replacing Ser with either Thr, Ala, or Gly possesses thelevel of activity disclosed above. According to Bowie et al. (Science247:1306-1310, 1990), if a particular property of a side chain, such ascharge or size, is important at a given position, only side chains thathave the required property will be allowed. Conversely, if the chemicalidentity of the side chain is unimportant, then many differentsubstitutions will be permitted. Studies based on these notions revealedthat proteins are surprisingly tolerant of amino acid substitutions(Bowie et al., supra at page 1306). Thus the art recognizes and acceptscertain types of changes in proteins and in peptides. Such acceptablemodifications delineate a genus of peptides wherein each speciespredictably has the requisite type and/or level of activity.

Further, Bordo and Argos, J. Mol. Biol. 217:721-729 (1991), reported astatistical analysis of protein sequences and provided guidelines for“safe” amino acid substitutions in protein design, and by analogy,peptide design. It is axiomatic that proteins with similar functions aretopographically similar at least in those regions responsible foractivity. Based on the fact that the peptide KPSSPPEE (SEQ ID NO: 2)contains 3 prolines out of 8 amino acids, the present inventorspredicted that this peptide would have a single major conformer insolution, perhaps differing only in proline isomerization. This was alsopredicted by molecular dynamics simulations, Hence, KPSSPPEE (SEQ ID NO:2) would not be expected to assume multiple conformers in solution. ThePP region of this peptide forms a specialized conformational motif knownas a “proline turn” (or “bend”). The stiffness of this peptide (and itspreliminary 3D structure) has been confirmed by 2D NMR.

In addition to applying this topographical criterion to the design andproduction of peptides with sequence homology or acceptable sequencesubstitutions, this criterion can be used as a basis for generatingchemical derivatives of KPSSPPEE (SEQ ID NO: 2), including thepeptidomimetics described above. This is fundamental to structure-baseddrug design and modeling. Although the solution structure of a freepeptide may not exactly mimic its bound conformation, the solutionstructure does provide a starting scaffold for optimizing derivativeswhich mimic the peptide's activity. In fact, such scaffolds could not bederived in the absence of the basic topographical information about thispeptide, either free or bound. If a derivative is prepared with astructure/topography similar to that of KPSSPPEE (SEQ ID NO: 2) and therequisite biological and binding activity as disclosed herein, then itis within the scope of the present invention.

If a peptide or peptidomimetic is designed in accordance with thisinvention based on either the sequence or the topography (structure) ofKPSSPPEE (SEQ ID NO: 2), and it has the bioactivity stated above, thenit must be similar in conformation to KPSSPPEE (SEQ ID NO: 2) andtherefore falls within the scope of the invention. The assessment ofactivity in bioassays or binding assays such as those described hereinis routine in the and is the logical way to determine whether a compoundis active. A useful substitution variant, addition variant or otherchemical derivative of KPSSPPEE (SEQ ID NO: 2) is a compound that hasbeen designed based on the sequence or topographical structure ofKPSSPPEE (SEQ ID NO: 2).

Systematic approaches in the art that allow the optimization of apeptide and development of peptidomimetics (Hruby et al., Biochem J.268:249-262 (1990); and Hruby, 1993, supra) flow from a single startingpoint: the identification of a peptide lead compound. For example, themost preferred peptide lead compound of this invention is KPSSPPEE (SEQID NO: 2). The peptide and peptidomimetic design approaches disclosedherein and/or known in the art for generating an optimized compound arenot possible without first identifying an active lead peptide, so thatthese designed peptides or peptidomimetics constitute a genus ofcompounds “around” the original peptide. Schemes for preparing activederivatives of the parent peptide have been described (e.g., Moore etal., Adv. Pharmacol. 33:91-141 (1995); Giannis and Rubsam, Adv. DrugResearch 29:1-78 (1997)). Although each approach may require someexperimentation, it is neither random nor undue. By following acceptedschemes practiced by those skilled in the art, one can generate familiesof similarly acting compounds.

Diagnostic and Prognostic Compositions

Further, the peptides can be labeled for detection and used, forexample, to detect a binding site for the peptide on the surface or inthe interior of a cell. Thus, the fate of the peptide can be followed invitro or in vivo by using the appropriate method to detect the label.The labeled peptide may also be utilized in vivo for diagnosis andprognosis, for example to image occult metastatic foci or for othertypes of in situ evaluations.

Example of suitable detectable labels are radioactive, fluorogenic,chromogenic, or other chemical labels. Useful radiolabels, which aredetected by a gamma counter or a scintillation counter or byautoradiography include ³H, ¹²⁵I, ¹³¹I ³⁵S and ¹⁴C. In addition, ¹³¹I isalso useful as a therapeutic isotope (see below).

Common fluorescent labels include fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

The fluorophore, such as the dansyl group, must be excited by light of aparticular wavelength to fluoresce. See, for example, Haugland, Handbookof Fluorescent Probes and Research Chemicals, Sixth Edition, MolecularProbes, Eugene, Oreg., 1996). In general, a fluorescent reagent isselected based on its ability to react readily with an amino function.Examples of such fluorescent probes include the Bodipy(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophores which span thevisible spectrum (U.S. Pat. No. 4,774,339; U.S. Pat. No. 5,187,288; U.S.Pat. No. 5,248,782; U.S. Pat. No. 5,274,113; U.S. Pat. No. 5,433,896;U.S. Pat. No. 5,451,663). A preferred member of this group is4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid.

Fluorescein, fluorescein derivatives and fluorescein-like molecules suchas Oregon Green™ and its derivatives, Rhodamine Green™ and RhodolGreen™, are coupled to amine groups using the isocyanate, succinimidylester or dichlorotriazinyl-reactive groups. The long wavelengthrhodamines, which are basically Rhodamine Green™ derivatives withsubstituents on the nitrogens, are among the most photostablefluorescent labeling reagents known. Their spectra are not affected bychanges in pH between 4 and 10, an important advantage over thefluoresceins for many biological applications. This group includes thetetramethylrhodamines, X-rhodanmines and Texas Red derivatives. Otherpreferred fluorophores for derivatizing the peptide according to thisinvention are those which are excited by ultraviolet light. Examplesinclude cascade blue, coumarin derivatives, naphthalenes (of whichdansyl chloride is a member), pyrenes and pyridyloxazole derivatives.

In yet another approach, one or more amino groups is allowed to reactwith reagents that yield fluorescent products, for example,fluorescamine, dialdehydes such as o-phthaldialdehyde,naphthalcne-2,3-dicarboxylate and anthracene-2,3-dicarboxylate.7-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives, both chloride andfluoride, are useful to modify amines to yield fluorescent products.

Those skilled in the art will recognize that known fluorescent reagentsmodify groups other than amines, such as thiols, alcohols, aldehydes,ketones, carboxylic acids and amides. Hence, fluorescent substrates canreadily be designed and synthesized using these other reactive groups.

The peptide can also be labeled for detection usingfluorescence-emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the peptide using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA). The peptide can be madedetectable by coupling it to a chemiluminescent compound. The presenceof the chemiluminescent-tagged peptide is then determined by detectingthe presence of luminescence that arises during the course of a chemicalreaction. Examples of particularly useful chemiluminescers are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester Likewise, a bioluminescent compound may be used to labelthe peptide. Bioluminescence is a type of chemiluminescence found inbiological systems in which a catalytic protein increases the efficiencyof the chemiluminescent reaction. The presence of a bioluminescentprotein is determined by detecting the presence of luminescence.Important bioluminescent compounds for purposes of labeling areluciferin, luciferase and aequorin.

In yet another embodiment, colorimetric detection is used, based onchromogenic compounds (chromophores) with high extinction coefficients.

In situ detection of the labeled peptide may be accomplished by removinga histological specimen from a subject and examining it by microscopyunder appropriate conditions to detect the label. Those of ordinaryskill will readily perceive that any of a wide variety of histologicalmethods (such as staining procedures) can be modified in order toachieve such in situ detection.

The term “diagnostically labeled” means that the peptide has attached toit a diagnostically detectable label. There are many different labelsand methods of labeling known to those of ordinary skill in the art.Examples of the types of labels which can be used in the presentinvention include radioactive isotopes, paramagnetic isotopes, andcompounds which can be imaged by positron emission tomography (PET).Those of ordinary skill in the art will know of other suitable labelsfor binding to the peptides used in the invention, or will be able toascertain such, by routine experimentation. Furthermore, the binding ofthese labels to the peptide or derivative can be done using standardtechniques known to those of ordinary skill in the art.

For diagnostic in vivo radioimaging, the type of detection instrumentavailable is a major factor in selecting a given radionuclide. Theradionuclide chosen must have a type of decay which is detectable by agiven type of instrument. In general, any conventional method forvisualizing diagnostic imaging can be utilized in accordance with thisinvention. Another factor in selecting a radionuclide for in vivodiagnosis is that the half-life of a radionuclide be long enough so thatit is still detectable at the time of maximum uptake by the targetissue, but short enough so that deleterious radiation of the host isminimized. In one preferred embodiment, a radionuclide used for in vivoimaging does not emit particles, but produces a large number of photonsin a 140-200 keV range, which may be readily detected by conventionalgamma cameras.

For in vivo diagnosis, radionuclides may be bound to peptide eitherdirectly or indirectly by using an intermediary functional group.Intermediary functional groups that are often used to bindradioisotopes, which exist as metallic ions, to peptides are thechelating agents, DTPA and EDTA. Examples of metallic ions which can bebound to peptides are ⁹⁹Tc, ¹²³I, ¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, ¹²⁵I,⁶⁸Ga, ⁷²As, ⁸⁹Zr, and ²⁰¹Tl. Generally, the dosage of peptide labeledfor detection for diagnostic use will vary depending on considerationssuch as age, condition, sex, and extent of disease in the patient,counterindications, if any, and other variables, to be adjusted by theindividual physician. Dosage can vary from 0.01 mg/kg to 100 mg/kg.

In another embodiment, the peptides or derivatives of the presentinvention are used as affinity ligands for binding the peptide'sreceptor in assays, preparative affinity chromatography or solid phaseseparation. Such compositions may also be used to enrich, purify orisolate cells to which the peptide or derivative binds, preferablythrough a specific receptor-ligand interaction. The peptide orderivative is immobilized using common methods known in the art, e.g.binding to CNBr-activated Sepharose® or Agarose®, NHS-Agarose® orSepharose®, epoxy-activated Sepharose® or Agarose®, EAH-Sepharose® orAgarose®, streptavidin-Sepharose® or Agarose® in conjunction withbiotinylated peptide or derivatives. In general the peptides orderivatives of the invention may be immobilized by any other methodwhich is capable of immobilizing these compounds to a solid phase forthe indicated purposes. See, for example Affinity Chromatography:Principles and Methods (Pharmacia LKB Biotechnology). Thus, oneembodiment is a composition comprising any of the peptides, derivativesor peptidomimetics described herein, bound to a solid support or aresin. The compound may be bound directly or via a spacer, preferably analiphatic chain having about 2-12 carbon atoms.

By “solid phase” or “solid support” or “carrier” is intended any supportor carrier capable of binding the peptide or derivative. Well-knownsupports, or carriers, in addition to Sepharose® or Agarose® describedabove are glass, polystyrene, polypropylene, polyethylene, dextran,nylon, amylases, natural and modified celluloses such as nitrocellulose,polyacrylamides, polyvinylidene difluoride, other agaroses, andmagnetite, including magnetic beads. The carrier can be totallyinsoluble or partially soluble. The support material may have anypossible structural configuration so long as the coupled molecule iscapable of binding to receptor material. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube or microplate well, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip,bottom surface of a microplate well, etc.

Antibodies and Their Uses

The present invention also provides antibodies specific for an epitopedefined by the peptide sequence KPSSPPEE (SEQ ID NO: 2) or specific fora chemical derivative thereof or a peptidomimetic thereof. Suchantibodies may be polyclonal, monoclonal, bispecific, chimeric orantiidiotypic, and include antigen-binding fragments thereof. Anyimmunoassay known in the art may be used to detect the binding of suchan antibody to a peptide, chemical derivative thereof or peptideoligomer according to this invention. Preferred assays are enzymeimmunoassays or radioimmunoassay. The following references (incorporatedby reference in their entirety) describe the production, purification,testing and use of antibodies: Hartlow, E. et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988; Campbell, A., In: Laboratory Techniques inBiochemistry and Molecular Biology, Volume 13 (Burdon, R., et al.,eds.), Elsevier, Amsterdam (1984)); Work, T. S. et al., LaboratoryTechniques and Biochemistry in Molecular Biology, North HollandPublishing Company, NY, 1978; Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986; Butler, J. E. (ed.),Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton, 1991;Butler, J. E., In: STRUCTURE OF ANTIGENS, Vol. 1, Van Regenmortel, M.,ed., CRC Press, Boca Raton 1992, pp. 209-259; Butler, J. E., In: vanOss, C. J. et al., (eds), IMMUNOCHEMISTRY, Marcel Dekker, Inc., NewYork, 1994, pp. 759-803; Voller, A. et al. (eds)., Immunoassays for the1980's, University Park Press, Baltimore, 1981.

Antibodies of this invention are used to detect the presence of ormeasure the amount of the peptide epitope in a biological material orother sample by direct or competitive immunoassay. The antibodies can becoupled to a solid support and used in affinity chromatography toisolate and purify material containing the peptide epitope. Conversely,as described above, the peptide, variant or chemical derivative of thisinvention, bound to a solid support, is used to enrich or purifyspecific antibodies. Antiidiotypic antibodies can be used to gain aknowledge of the structure of a peptide, variant or chemical derivativeof this invention when bound to a receptor for it.

Biological Assay of Anti-Invasive Activity

The compositions of the invention are tested for their anti-invasivecapacity in a Matrigel® invasion assay system as described in detail byKleinman et al., 1986 and Parish et al., 1992, which references arehereby incorporated by reference in their entirety. The assay isperformed with a cell line, more preferably a tumor cell line, mostpreferably the rat breast cancer (Mat BIII) line or the human prostatecancer (PC-3) line (Xing and Rabbani, 1996; Hoosein et al., 1991).

Matrigel® is a reconstituted basement membrane containing type IVcollagen, laminim, heparan sulfate proteoglycans such as perlecan, whichbind to and localize bFGF, vitronectin as well as transforming growthfactor-β (TGFβ), urokinase-type plasminogen activator (uPA), tissueplasminogen activator (tPA), and the serpin known as plasminogenactivator inhibitor type 1 (PAI-1) (Chambers et al., 1995).

It is accepted in the art that results obtained in this assay forcompounds which target extracellular receptors or enzymes are predictiveof the efficacy of these compounds in vivo (Rabbani et al., 1995).

Biological Assay of Anti-Angiogenic Activity

The compounds of this invention are tested for their anti-angiogenicactivity in one of two different assay systems in vitro.

Endothelial cells, for example, human umbilical vein endothelial cells(HUVEC) or human microvascular endothelial cells (HMVEC) which can beprepared or obtained commercially, are mixed at a concentration of 2×10⁵cells/mL with fibrinogen (5 mg/mL in phosphate buffered saline (PBS) ina 1:1 (v/v) ratio. Thrombin is added (5 units/mL final concentration)and the mixture is immediately transferred to a 24-well plate (0.5 mLper well). The fibrin gel is allowed to form and then VEGF and bFGF areadded to the wells (each at 5 ng/mL final concentration) along with thetest compound. The cells are incubated at 37° C. in 5% CO₂, for 4 daysat which time the cells in each well are counted and classified aseither rounded, elongated with no branches, elongated with one branch,or elongated with 2 or more branches. Results are expressed as theaverage of 5 different wells for each concentration of compound.Typically, in the presence of angiogenic inhibitors, cells remain eitherrounded or form undifferentiated tubes (e.g. 0 or 1 branch).

This assay is recognized in the art to be predictive of angiogenic (oranti-angiogenic) efficacy in vivo (Min et al., 1996).

In an alternate assay, endothelial cell tube formation is observed whenendothelial cells are cultured on Matrigel® (Schnaper et al., 1995).Endothelial cells (1×10⁴ cells/well) are transferred ontoMatrigel®-coated 24-well plates, and tube formation is quantitated after48 hrs. Inhibitors are tested by adding them either at the same time asthe endothelial cells or at various time points thereafter.

This assay models angiogenesis by presenting to the endothelial cells aparticular type of basement membrane, namely the layer of matrix whichmigrating and differentiating endothelial cells might be expected tofirst encounter. In addition to bound growth factors, the matrixcomponents found in Matrigel® (and in basement membranes in situ) orproteolytic products thereof may also be stimulatory for endothelialcell tube formation which makes this model complementary to the fibringel angiogenesis model previously described (Blood and Zetter, 1990;Odedra and Weiss, 1991). The compounds of this invention inhibitendothelial cell tube formation in both assays, which suggests that thecompounds will also have anti-angiogenic activity.

In Vivo Testing of Compositions in Animal Models of Human Tumors

The peptides, peptidomimetics and conjugates are tested for therapeuticefficacy in several well established rodent models which are consideredto be highly representative of a broad spectrum of human tumors. Theapproaches are described in detail in Geran, R. I. et al., “Protocolsfor Screening Chemical Agents and Natural Products Against Animal Tumorsand Other Biological Systems (Third Edition)”, Canc. Chemother. Reports,Part 3, 3:1-112, which is hereby incorporated by reference in itsentirety. All general test evaluation procedures, measurements andcalculations are performed in accordance with this reference, includingmean survival time, median survival time, calculation of approximatetumor weight from measurement of tumor diameters with vernier calipers;calculation of tumor diameters; calculation of mean tumor weight fromindividual excised tumors; and ratios between treated and control groupsratio for any measure (T/C ratios).

A. Rat Model of Tumor Progression

The effects of the compounds are tested on tumor progression in a ratsyngeneic model of breast cancer (Xing and Rabbani, 1996). Mat BIII ratbreast tumor cells (1×10⁶ cells in PBS, 0.1 mL per rat) are inoculatedinto the mammary fat pads of female Fisher rats. The test compound isdissolved in PBS (200 mM stock), sterile filtered and dispensed in vivoat a dose of up to about 100 mg/kg/day) using a 14-day Alza osmoticmini-pump implanted intraperitoneally at the time of inoculation.Control animals receive vehicle (PBS) alone. Animals are euthanized atday 14 and examined for metastasis in the spleen, lungs, liver, kidneyand lymph nodes. In addition, the primary tumors are excised,quantitated, and prepared for immunohistochemistry.

B. 3LL Lewis Lung Carcinoma: Primary Tumor Growth

This tumor line arose spontaneously in 1951 as carcinoma of the lung ina C57BL/6 mouse (Cancer Res 15:39, 1955. See, also Malave, I. et al., J.Nat'l. Canc Inst. 62:83-88 (1979)). It is propogated by passage inC57BL/6 mice by subcutaneous (sc) inoculation and is tested insemiallogeneic C57BL/6×DBA/2 F₁ mice or in allogeneic C3H mice.Typically six animals per group for subcutaneously (sc) implant, or tenfor intramuscular (im) implant are used. Tumor may be implanted sc as a2-4 mm fragment, or im or sc as an inoculum of suspended cells of about0.5-2×10⁶-cells. Treatment begins 24 hours after implant or is delayeduntil a tumor of specified size (usually approximately 400 mg) can bepalpated. The test compound is administered ip daily for 11 days

Animals are followed by weighing, palpation, and measurement of tumorsize. Typical tumor weight in untreated control recipients on day 12after iminoculation is 500-2500 mg. Typical median survival time is18-28 days. A positive control compound, for example cyclophosphamide at20 mg/kg/injection per day on days 1-11 is used. Results computedinclude mean animal weight, tumor size, tumor weight, survival time Forconfirmed therapeutic activity, the test composition should be tested intwo multi-dose assays.

C. 3LL Lewis Lung Carcinoma: Primary Growth and Metastasis Model

This model has been utilized by a number of investigators. See, forexample, Gorelik, E. et al., J. Nat'l. Canc. Inst. 65:1257-1264 (1980);Gorelik, E. et al., Rec. Results Canc Res. 75:20-28 (1980); Isakov, N.et al., Invasion Metas. 2:12-32 (1982); Talmadge J. E. et al., J. Nat'l.Canc. Inst. 69:975-980 (1982); Hilgard, P. et al, Br. J. Cancer 35:78-86(1977)). Test mice are male C57BL/6 mice, 2-3 months old. Following sc,im, or intra-footpad implantation, this tumor produces metastases,preferentially in the lungs. With some lines of the tumor, the primarytumor exerts anti-metastatic effects and must first be excised beforestudy of the metastatic phase (see also U.S. Pat. No. 5,639,725).

Single-cell suspensions are prepared from solid tumors by treatingminced tumor tissue with a solution of 0.3% trypsin. Cells are washed 3times with PBS (pH 7.4) and suspended in PBS. Viability of the 3LL cellsprepared in this way is generally about 95-99% (by trypan blue dyeexclusion). Viable tumor cells (3×10⁴-5×10⁶) suspended in 0.05 ml PBSare injected subcutaneously, either in the dorsal region or into onehind foot pad of C57BL/6 mice. Visible tumors appear after 3-4 daysafter dorsal sc injection of 10⁶ cells. The day of tumor appearance andthe diameters of established tumors are measured by caliper every twodays.

The treatment is given as one or two doses of peptide or derivative, perweek. In another embodiment, the peptide is delivered by osmoticminipump.

In experiments involving tumor excision of dorsal tumors, when tumorsreach about 1500 mm³ in size, mice are randomized into two groups: (1)primary tumor is completely excised; or (2) sham surgery is performedand the tumor is left intact. Although tumors from 500-3000 mm³ inhibitgrowth of metastases, 1500 mm³ is the largest size primary tumor thatcan be safely resected with high survival and without local regrowth.After 21 days, all mice are sacrificed and autopsied.

Lungs are removed and weighed. Lungs are fixed in Bouin's solution andthe number of visible metastases is recorded. The diameters of themetastases are also measured using a binocular stereoscope equipped witha micrometer-containing ocular under 8× magnification. On the basis ofthe recorded diameters, it is possible to calculate the volume of eachmetastasis. To determine the total volume of metastases per lung, themean number of visible metastases is multiplied by the mean volume ofmetastases. To further determine metastatic growth, it is possible tomeasure incorporation of ¹²⁵IdUrd into lung cells (Thakur, M. L. et al.,J. Lab. Clin. Med. 89:217-228 (1977). Ten days following tumoramputation, 25 μg of fluorodeoxyuridine is inoculated into theperitoneums of tumor-bearing (and, if used, tumor-resected mice). After30 min, mice are given 1 μCi of ¹²⁵IdUrd (iododeoxyuridine). One daylater, lungs and spleens are removed and weighed, and a degree of¹²⁵IdUrd incorporation is measured using a gamma counter.

In mice with footpad tumors, when tumors reach about 8-10 mm indiameter, mice are randomized into two groups: (1) legs with tumors areamputated after ligation above the knee joints; or (2) mice are leftintact as nonamputated tumor-bearing controls. (Amputation of atumor-free leg in a tumor-bearing mouse has no known effect onsubsequent metastasis, ruling out possible effects of anesthesia, stressor surgery). Mice are killed 10-14 days after amputation. Metastases areevaluated as described above.

Statistics: Values representing the incidence of metastases and theirgrowth in the lungs of tumor-bearing mice are not normally distributed.Therefore, non-parametric statistics such as the Mann-Whitney U-Test maybe used for analysis.

Study of this model by Gorelik et al. (1980, supra) showed that the sizeof the tumor cell inoculum determined the extent of metastatic growth.The rate of metastasis in the lungs of operated mice was different fromprimary tumor-bearing mice. Thus in the lungs of mice in which theprimary tumor had been induced by inoculation of larger doses of 3LLcells (1-5×10⁶) followed by surgical removal, the number of metastaseswas lower than that in nonoperated tumor-bearing mice, though the volumeof metastases was higher than in the nonoperated controls. Using¹²⁵IdUrd incorporation as a measure of lung metastasis, no significantdifferences were found between the lungs of tumor-excised mice andtumor-bearing mice originally inoculated with 1×10⁶ 3LL cells.Amputation of tumors produced following inoculation of 1×10⁵ tumor cellsdramatically accelerated metastatic growth. These results were in accordwith the survival of mice after excision of local tumors. The phenomenonof acceleration of metastatic growth following excision of local tumorshad been repeatedly observed (for example, see U.S. Pat. No. 5,639,725).These observations have implications for the prognosis of patients whoundergo cancer surgery.

D. Experimental Metastasis Models

The compounds of this invention are also tested for inhibition of latemetastasis using an experimental metastasis model (Crowley et al.,1993). Late metastasis involves the steps of attachment andextravasation of tumor cells, local invasion, seeding, proliferation andangiogenesis.

Human prostatic carcinoma cells (PC-3) transfected with a reporter gene,preferably the green fluorescent protein (GFP) gene, but as analternative with a gene encoding the enzymes chioramphenicolacetyl-transferase (CAT), luciferase or LacZ. This permits utilizationof either of these markers (fluorescence detection of GFP orhistochemical colorimetric detection of enzymatic activity) forfollowing the fate of these cells. Cells are injected, preferably iv,and metastases identified after about 14 days, particularly in the lungsbut also in regional lymph nodes, femurs and brain. This mimics theorgan tropism of naturally occurring metastases of prostate cancer. Forexample, GFP-expressing PC-3 cells (1×10⁶ cells per mouse) are injectediv into the tail veins of nude (nu/nu) mice. Animals are also implantedwith mini-pumps (sub-dermally on the back) dispensing either the testcompound (at least about 100 mg/kg/day) or vehicle. The animals areeuthanized after 14 days and their organs prepared for histologicalexamination. Single metastatic cells and foci are visualized andquantitated by fluorescence microscopy or light microscopichistochemistry or by grinding the tissue and quantitative colorimetricassay of the detectable label.

For a compound to be useful in accordance with this invention, it shoulddemonstrate anti-tumor activity in the above models, for example,blocking tumor progression, angiogenesis and/or metastasis.

Angiogenesis

Angiogenesis is measured by determining microvessel density usingimmunostaining for CD31 (also known as platelet-endothelial celladhesion molecule or PECAM). Results are reported as the averagemicrovessel density of 5 fields each from 5 different sections (Penfoldet al., 1996). Typically, the whole tumor is excised, sectioned and thesections examined histologically for microvessel density usingappropriate stains or labels for other markers.

Pharmaceutical and Therapeutic Compositions and Their Administration

The compounds that may be employed in the pharmaceutical compositions ofthe invention include all of those compounds described above, as well asthe pharmaceutically acceptable salts of these compounds.Pharmaceutically acceptable acid addition salts of the compounds of theinvention containing a basic group are formed where appropriate withstrong or moderately strong, non-toxic, organic or inorganic acids inthe presence of a basic amine by methods known to the art. Exemplary ofthe acid addition salts that are included in this invention are maleate,fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate,benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide,sulfate, phosphate and nitrate salts.

Pharmaceutically acceptable base addition salts of compounds of theinvention containing an acidic group are prepared by known methods fromorganic and inorganic bases and include, for example, nontoxic alkalimetal and alkaline earth bases, such as calcium, sodium, potassium andammonium hydroxide; and nontoxic organic bases such as triethylamine,butylamine, piperazine, and tri(hydroxymethyl)methylamine.

As stated above, the compounds of the invention possess the ability toinhibit invasiveness or angiogenesis, properties that are exploited inthe treatment of cancer, in particular metastatic cancer. A compositionof this invention may be active per se, or may act as a “pro-drug” thatis converted in vivo to the active form.

The compounds of the invention, as well as the pharmaceuticallyacceptable salts thereof, may be incorporated into convenient dosageforms, such as capsules, impregnated wafers, tablets or injectablepreparations. Solid or liquid pharmaceutically acceptable carriers maybe employed.

Preferably, the compounds of the invention are administeredsystemically, e.g., by injection. When used, injection may be by anyknown route, preferably intravenous, subcutaneous, intramuscular,intracranial or intraperitoneal. Injectables can be prepared inconventional forms, either as solutions or suspensions, solid formssuitable for solution or suspension in liquid prior to injection, or asemulsions.

Solid carriers include starch, lactose, calcium sulfate dihydrate, terraalba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearateand stearic acid. Liquid carriers include syrup, peanut oil, olive oil,saline, water, dextrose, glycerol and the like. Similarly, the carrieror diluent may include any prolonged release material, such as glycerylmonostearate or glyceryl distearate, alone or with a wax. When a liquidcarrier is used, the preparation may be in the form of a syrup, elixir,emulsion, soft gelatin capsule, sterile injectable liquid (e.g., asolution), such as an ampoule, or an aqueous or nonaqueous liquidsuspension. A summary of such pharmaceutical compositions may be found,for example, in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton Pa. (Gennaro 18th ed. 1990).

The pharmaceutical preparations are made following conventionaltechniques of pharmaceutical chemistry involving such steps as mixing,granulating and compressing, when necessary for tablet forms, or mixing,filling and dissolving the ingredients, as appropriate, to give thedesired products for oral, parenteral, topical, transdermal,intravaginal, intranasal, intrabronchial, intracranial, intraocular,intraaural and rectal administration. The pharmaceutical compositionsmay also contain minor amounts of nontoxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents and so forth.

Though the preferred routes of administration are systemic thepharmaceutical composition may be administered topically ortransdermally, e.g., as an ointment, cream or gel; orally; rectally;e.g., as a suppository, parenterally, by injection or continuously byinfusion; intravaginally; intranasally; intrabronchially; intracraniallyintra-aurally; or intraocularly.

For topical application, the compound may be incorporated into topicallyapplied vehicles such as a salve or ointment. The carrier for the activeingredient may be either in sprayable or nonsprayable form.Non-sprayable forms can be semi-solid or solid forms comprising acarrier indigenous to topical application and having a dynamic viscositypreferably greater than that of water. Suitable formulations include,but are not limited to, solution, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like. If desired, thesemay be sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, wetting agents, buffers, or salts for influencing osmoticpressure and the like. Preferred vehicles for non-sprayable topicalpreparations include ointment bases, e.g., polyethylene glycol-1000(PEG-1000); conventional creams such as HEB cream; gels; as well aspetroleum jelly and the like.

Also suitable for topic application are sprayable aerosol preparationswherein the compound, preferably in combination with a solid or liquidinert carrier material, is packaged in a squeeze bottle or in admixturewith a pressurized volatile, normally gaseous propellant. The aerosolpreparations can contain solvents, buffers, surfactants, perfumes,and/or antioxidants in addition to the compounds of the invention.

For the preferred topical applications, especially for humans, it ispreferred to administer an effective amount of the compound to aninfected area, e.g., skin surface, mucous membrane, eyes, etc. Thisamount will generally range from about 0.001 mg to about 1 g perapplication, depending upon the area to be treated, the severity of thesymptoms, and the nature of the topical vehicle employed.

The compositions of the invention may further comprise one or moreadditional compounds that are anti-tumor agents, such as mitoticinhibitors, e.g., vinblastine; alkylating agents, e.g.,cyclophosphamide; folate inhibitors, e.g., methotrexate, piritrexim ortrimetrexate; antimetabolites, e.g., 5-fluorouracil and cytosinearabinoside; intercalating antibiotics, e.g., adriamycin and bleomycin;enzymes or enzyme inhibitors, e.g., asparaginase; topoisomeraseinhibitors, e.g., etoposide; or biological response modifiers, e.g.,interferon. In fact, pharmaceutical compositions comprising any knowncancer therapeutic in combination with the peptides disclosed herein arewithin the scope of this invention.

The composition of the invention may also comprise one or more othermedicaments, preferably anti-infectives such as antibacterial,anti-fungal, anti-parasitic, anti-viral, and anti-coccidial agents.Exemplary antibacterial agents include, for example, sulfonamides suchas sulfamethoxazole, sulfadiazine or sulfadoxine; DIFR inhibitors suchas Htrimethoprim, bromodiaprim or trimetrexate; penicillins;cephalosporins; aminoglycosides; bacteriostatic inhibitors of proteinsynthesis; the quinolonecarboxylic acids and their fused isothiazoleanalogs; and the like.

Other Therapeutic Compositions

In another embodiment, the compounds of this invention are“therapeutically conjugated” and used to deliver a therapeutic agent tothe site of where the compounds home and bind, such as sites of tumormetastasis or foci of infection/inflammation. The term “therapeuticallyconjugated” means that the compound, preferably a peptide, peptidederivative, or peptidomimetic, is conjugated to a therapeutic agent. Thetherapeutic agents used in this manner act are directed either to theunderlying cause or the components of the processes of tumor invasion,angiogenesis or inflammation. Examples of agents used to treatinflammation are the steroidal and non-steroidal anti-inflammatorydrugs, many of which inhibit prostaglandin synthesis.

Other therapeutic agents which can be coupled to the compounds accordingto the method of the invention are drugs, radioisotopes, lectins andother toxins. The therapeutic dosage administered is an amount which istherapeutically effective, and will be known to one of skill in the art.The dose is also dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired, such as, for example,anti-inflammatory effects or anti-bacterial effect.

Lectins are proteins, commonly derived from plants, that bind tocarbohydrates. Among other activities, some lectins are toxic. Some ofthe most cytotoxic substances known are protein toxins of bacterial andplant origin (Frankel, A. E. et al., Ann. Rev. Med. 37:125-142 (1986)).These molecules binding the cell surface and inhibition cellular proteinsynthesis. The most commonly used plant toxins are ricin and abrin; themost commonly used bacterial toxins are diphtheria toxin and Pseudomonasexotoxin A. In ricin and abrin, the binding and toxic functions arecontained in two separate protein subunits, the A and B chains. Thericin B chain binds to the cell surface carbohydrates and promotes theuptake of the A chain into the cell. Once inside the cell, the ricin Achain inhibits protein synthesis by inactivating the 60S subunit of theeukaryotic ribosome Endo, Y. et al., J. Biol. Chem. 262: 5908-5912(1987)). Other plant derived toxins, which are single chain ribosomalinhibitory proteins, include pokeweed antiviral protein, wheat germprotein, gelonin, dianthins, momorcharins, trichosanthin, and manyothers (Strip, F. et al., FEBS Lett. 195:1-8 (1986)). Diphtheria toxinand Pseudomonas exotoxin A are also single chain proteins, and theirbinding and toxicity functions reside in separate domains of the sameprotein chain with full toxin activity requiring proteolytic cleavagebetween the two domains. Pseudomonas exotoxin A has the same catalyticactivity as diphtheria toxin. Ricin has been used therapeutically bybinding its toxic α-chain, to targeting molecules such as antibodies toenable site-specific delivery of the toxic effect. Bacterial toxins havealso been used as anti-tumor conjugates. As intended herein, a toxicpeptide chain or domain is bound to a compound of this invention anddelivered in a site-specific manner to a target site where the toxicactivity is desired, such as a metastatic focus. Conjugation of toxinsto protein such as antibodies or other ligands are known in the art(Olsnes, S. et al., Immunol. today 10:291-295 (1989); Vitetta, E. S. etal., Ann. Rev. Immunol. 3:197-212 (1985)).

Examples of therapeutic radioisotopes which can be bound to the compoundfor use in accordance with according the methods of the invention are¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Bi, ²¹¹At, ²¹²Pb, ⁴⁷Sc, and ¹⁰⁹Pd.

Cytotoxic drugs that interfere with critical cellular processesincluding DNA, RNA, and protein synthesis, have been conjugated toantibodies and subsequently used for in vivo therapy. Such drugs,including but are not limited to daunorubicin, doxorubicin,methotrexate, and Mitomycin C are also coupled to the compounds of thisinvention and use therapeutically in this form.

Therapeutic Methods

This invention includes methods for inhibiting cellular invasion,chiefly by tumor cells, or angiogenesis, primarily induced by tumorcells in a subject. By inhibiting invasion by cells or angiogenesis, themethod results in inhibition of tumor metastasis. In this method, avertebrate subject, preferably a mammal, more preferably a human, isadministered an amount of the compound effective to inhibit invasion orangiogenesis. The compound or pharmaceutically acceptable salt thereofis preferably administered in the form of a pharmaceutical compositionas described above.

Doses of the compounds preferably include pharmaceutical dosage unitscomprising an effective amount of the peptide. By an effective amount ismeant an amount sufficient to achieve a steady state concentration invivo which results in a measurable reduction in any relevant parameterof disease and may include growth of primary or metastatic tumor, anyaccepted index of inflammatory reactivity, or a measurable prolongationof disease-free interval or of survival. For example, a reduction intumor growth in 20% of patients is considered efficacious (Frei I I I,E., The Cancer Journal 3:127-13b (1997)). However, an effect of thismagnitude is not considered to be a minimal requirement for the dose tobe effective in accordance with this invention.

In one embodiment, an effective dose is at least equal to, preferably10-fold and more preferably 100-fold higher than the 50% inhibitoryconcentration (IC₅₀) of the compound in an in vivo assay as describedherein.

The amount of active compound to be administered depends on the precisepeptide or derivative selected, the disease or condition, the route ofadministration, the health and weight of the recipient, the existence ofother concurrent treatment, if any, the frequency of treatment, thenature of the effect desired, for example, inhibition of tumormetastasis, and the judgment of the skilled practitioner.

A preferred dose for treating a subject, preferably mammalian, morepreferably human, with a tumor is an amount of up to about 100milligrams of active compound per kilogram of body weight.

Typical single dosages of the peptide are between about I μg and about100 mg/kg body weight. For topical administration, dosages in the rangeof about 0.01-20% concentration of the compound, preferably 1-5%, aresuggested. A total daily dosage in the range of about 10 milligrams toabout 7 grams is preferred for oral administration. The foregoing rangesare, however, suggestive, as the number of variables in regard to anindividual treatment regime is large, and considerable excursions fromthese recommended values are expected.

An effective amount or dose of the peptide for inhibiting invasion invitro is in the range of about 1 picogram to about 0.5 nanograms percell. Effective doses and optimal dose ranges may be determined in vitrousing the methods described herein.

The compounds of the invention may be further characterized as producingan inhibitory effect on cell migration and invasion, on angiogenesis, ontumor metastasis or on inflammatory reactions. The compounds areespecially useful in producing an anti-tumor effect in a mammalian host,preferably human, harboring a tumor.

The foregoing compositions and treatment methods are useful forinhibiting cell migration and invasion or migration-induced cellproliferation in a subject having a disease or condition associated withundesired cell invasion, migration-induced proliferation, angiogenesisor metastasis. Such diseases or conditions may include primary growth orsolid tumors or leukemias and lymphomas, metastasis, invasion and/orgrowth of tumor metastases, atherosclerosis, myocardial angiogenesis,post-balloon angioplasty vascular restenosis, neointima formationfollowing vascular trauma, vascular graft restenosis, coronarycollateral formation, deep venous thrombosis, ischemic limbangiogenesis, telangiectasia, pyogenic granuloma, corneal diseases,rubeosis, neovascular glaucoma, diabetic and other retinopathy,retrolental fibroplasia, diabetic neovascularization, maculardegeneration, endometriosis, arthritis, fibrosis associated with chronicinflammatory conditions including psoriasis scleroderma, lung fibrosis,chemotherapy-induced fibrosis, wound healing with scarring and fibrosis;peptic ulcers, fractures, keloids, and disorders of vasculogenesis,hematopoiesis, ovulation, menstruation, pregnancy and placentation, orany other disease or condition in which invasion or angiogenesis ispathogenic.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

Example I Synthesis of Acetyl-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-NH₂ (SEQID NO: 2)

The starting material was p-methyl-benzhydrylamine resin substituted ata level of 0.70 mEq per gram of resin. Each of the L-amino acids,starting with glutamic acid, was added in sequence in a synthesis cycleconsisting of the three steps of TFA deprotection, coupling and capping.The completed peptide was subjected to HF cleavage and then purified.

1. TFA De-Protection

The starting resin was conditioned before adding the first glutamicacid, or, in the case of subsequent cycles, the BOC protecting group wasremoved from the α-amino nitrogen of the starting material by treatingthe resin with 50% trifluoroacetic acid (TFA) in dichloromethane (DCM)(two to three volumes per resin volume). The mixture was stirred at roomtemperature for 30 minutes and then drained. The resin was then washedonce with an equal volume of isopropanol for one minute and washed twicewith an equal volume of methanol, each wash taking one minute.

2. Coupling

The de-protected resin was washed twice with an equal volume of 10%triethylamine in DCM, each wash taking one minute, and washed twice withan equal volume of methanol, each wash taking one minute, and washedtwice with an equal volume of DCM, each wash taking one minute. ABOC-protected amino acid (three equivalents, dissolved in DCM or in amixture of DCM and N,N′-dimethylformamide (DMF)) and1-hydroxybenzotriazole (1M solution in DMF, three equivalents) was addedto the resin, and the mixture was stirred for a few seconds.Dicyclohexylcarbodiimide (DCC) (1M solution in DCM, three equivalents)was then added, and the whole mixture was stirred for 60-120 minutes.The resin was washed twice with an equal volume of methanol and thenwashed twice with an equal volume of DCM. A small sample was taken for aninhydrin test to assess the completeness of coupling. Generally, ifincomplete, the coupling step 2 is repeated. If complete, the synthesisis continued with the capping step 3.

All amino acids were used as α-BOC derivatives. Side chain protectinggroups were as follows:

Amino acid Protecting group Histidine Benzyloxymethyl Asparagine XanthylGlutamine Xanthyl Serine O-benzyl Theconine O-benzyl Tyrosine 2-Bromo-ZLysine 2-Chloro-Z Glutamic acid Cyclohexyl Aspartic acid Cyclohexyl

3. Capping

The resin was stirred with an equal volume of acetic anhydride (20%solution in DCM) for 5 minutes at room temperature. The resin was washedtwice with an equal volume of methanol and then washed twice with anequal volume of DCM.

4. HF Cleavage

The resin bearing the desired amino acid sequence (1.0 gram) was placedin a Teflon reaction vessel, and anhydrous anisole (1 mL) was added. Thevessel was cooled with liquid N₂, and anhydrous HF (10 mL) was distilledinto it. The temperature was raised with ice water to 0° C. The mixturewas stirred at this temperature for one hour, and then the HF wasdistilled off at 0° C. The residue was washed with anhydrous ether, andthe peptide was extracted with a 1:1 mixture of CH₃CN:H₂0.

5. Purification

The lyophilized powder was dissolved in 0.1% TFA buffer and loaded ontoa Waters C18 preparative column (2 inches diameter, 15-20 μm particlesize, 300 Å pore size). The loaded column was eluted with atwo-component eluent applied as a linear gradient, starting with 0% ofsolution A in solution B and finishing with 40% of solution A insolution B. Solution A was 0.1% TFA in H₂O, and solution B was 0.1% TFAin CH₃CN. Fractions exhibiting purity equal to or better than thatdesired were pooled and lyophilized to render the purified final productas the trifluoroacetate salt.

Example II Anti-Invasive and Anti-Proliferative Activity of CappedKPSSPPEE (SEQ ID NO: 2) (Å6) and Related Peptides

Several peptides were tested for anti-invasive capacity in a Matrigel®invasion assay system as indicated above (Kleinman et al., supra; Parishet al., supra). Several invasive human tumor lines (PC-3, MDA-MB-231)and non-human tumors (3LL, Mat B-III) were examined. The rat breastcancer line Mat B III and the human prostate cancer line PC-3 wereinitially used.

Tumor cells (5×10⁵/mL, in a volume of 200 μL) in serum-free RPMI 1640medium were added to a disposable transwell invasion chamber coated withMatrigel® (Becton Dickinson, Lincoln Park, N.J.). The invasion chamberswere placed in 24-well tissue culture plates filled with serum-freeRPMI-1640 and the plates were placed in an atmosphere of 5% CO₂ inhumidified air at 37° C. for 48-72 hours. The chambers were thenremoved, inverted, and the cells which had invaded (and now appeared onthe bottom face of the invasion chamber) were fixed and stained usingDiff-Quick® (Scientific Products). Cells were counted in 10 differentfields on each filter and an average obtained. Typically, 3-5 replicateswere performed at each concentration of compound tested.

The invasion of all the cell lines tested thus far has been inhibited byÅ6. FIG. 1 shows that Ac-KPSSPPEE-Am (SEQ ID NO: 2) inhibited theinvasion of both rat and human prostate cancer line PC-3 and rat breastcancer line Mat B III. Typical results with both MDAMB-231 and Mat B-IIIcells are presented in FIG. 7.

This peptide was not cytotoxic to the cells nor did it inhibit cellproliferation. Thus the observed effect was not a side effect ofcytotoxicity and could be ascribed to a mechanism of action distinctfrom that of cytotoxic or cytostatic agents.

Tests were also conducted on shorter related, capped peptides having thesequence Ac-PSSPPEE-Am (SEQ ID NO: 4) (a deletion variant of SEQ ID NO:2which lacks the N-terminal Lys) and Ac-KPSSPPE-Am (SEQ ID NO: 5) (adeletion variant of SEQ ID NO:2 lacking one of the C-terminal Gluresidues). Also tested was a similar longer peptide, KPSSPPEELK [SEQ IDNO: 1] (Blasi et al., U.S. Pat. No. 5,416,006) and its cappedcounterpart, Ac-KPSSPPEELK-Am) (SEQ ID NO: 1).

To identify the minimal sequence required for activity as well as toassess the role of the capping group for activity, Å6 and variants of Å6were tested for their ability to inhibit Mat B-III invasion throughMatrigel (FIG. 8). Å6 was found to be optimal.

It is noteworthy that all the peptides other than Ac-KPSSPPEE-Am (SEQ IDNO: 2) showed little or no activity in this assay, indicating that SEQID NO: 2 was the minimal required size for activity (FIG. 2). Theresults also indicated that addition of Leu and Lys at the C terminus ofKPSSPPEE (SEQ ID NO: 2) abrogated its biological activity, regardless ofwhether the termini were capped or uncapped.

The anti-invasive and anti-migratory properties of prompted theinventors to test whether Å6 inhibited the matrix metalloproteinasesMMP2 and MMP9. HT1080 cells were grown in the presence of dexamethasonefor 24 hrs and the supernatant collected and treated with phenylmercuricacetate to activate proMMPs. MMP activity was measured using theEnzCheck gelatinase assay (Molecular Probes). Å6 did not inhibitgelatinase activity at concentrations up to 100 μM.

Å6 was also tested for its ability to inhibit the proliferation of cellsother than endothelial cells in vitro. No anti-proliferative effectswere observed when Å6 was tested against U87, MDA-MB-231, Mat B-III,HeLa, CHO, HepG2 or SMC (aortic smooth muscle cells). Further, Å6 didnot potentiate the anti-proliferative activity of CDDP against U87cells.

Example III Inhibition of Plasminogen Activation

Single chain uPA (scuPA) complexed with a soluble form (suPAR) of theuPA receptor (uPAR) is able to activate plasminogen as efficiently asuPA, in the absence of activation by plasmin (Higazi A. A. R. et al.,(1995) J Biol Chem 270: 17375-17380). scuPA remains as a single chainmolecule, yet complex formation with suPAR induces a conformationalchange in scuPA, such that an active site capable of activatingplasminogen was formed. This scuPA-suPAR complex mimics the scuPA-uPARcomplex formed on the cell surface and activation of plasminogen byscuPA bound to cell-surface uPAR has indeed been demonstrated (ManchandaN. et al., (1991) J Biol chem. 266: 12752-12758). In addition, thescuPA-suPAR complex has been demonstrated to mediate clot lysis (fibrinturnover) in vitro and was more efficient in this assay than uPA alone.The scuPA-uPAR (or scuPA-suPAR) complex is very resistant to inhibitionby endogenous uPA inhibitors (PAIs) (Higazi, A R, Mazar A et al. (1996)Blood 87:3545-3549), in contrast to uPA, which is rapidly quenched inthe presence of PAIs.

Since Å6 was originally derived from uPA, its ability to inhibit variousactivities of uPA were tested, including binding to uPAR and theactivation of plasminogen. Å6 had no effect in any of these assaysexcept that it inhibited scuPA-suPAR mediated clot lysis (FIG. 3).

Å6 did not affect the activation of plasminogen by scuPA-suPAR whensmall, chromogenic substrates were used, and it did not affect theactivity of either uPA or plasmin directly. The requirement of proteinco-factors for the activation of plasminogen has been demonstrated by(Higazi et al., (1998) Blood 92, 2075-2083). Å6 may affect formation ofa tertiary or ternary complex required for the activation of plasminogenin the clot lysis assay.

Example IV Actions of Å6 on Endothelial Cells

Å6 was tested for its ability to inhibit HUVEC proliferation. Noinhibition was observed at concentrations as high as 312 μg/mL (340 μM)when HUVEC were grown on gelatin in 2% FBS (FIG. 4). Å6 did notpotentiate the anti-proliferative activity of cisplatin (CDDP) againstendothelial cells (FIG. 5).

The ability of Å6 to inhibit endothelial cell migration was evaluatedusing HUVEC and lung and dermal microvessel endothelial cells (HMVEC)with essentially identical results. Typical results are presented inFIG. 6.

Example V Receptor Studies

The present inventors prepared Å6-biotin (conjugated to the N-terminus)and used this as a probe for receptor binding and identification. Thisconjugate was cross-linked to whole cells and cell extracts in anattempt to identify candidate receptors for Å6. After cross-linking, theextracts were resolved by SDS-PAGE and transferred to PVDF membranes.Cross-linked products were detected using streptavidin-HRPO. Afteranalyzing the cross-linked products from several cell line extracts, theonly candidate product thus far identified corresponds to a molecularweight of about 30 kDa (FIG. 9). Further, this product seems to bepresent only in the detergent phase of the extract, suggesting that itis membrane bound. The more intensely staining high-molecular weightproducts likely bind biotin directly, as they could not be competed withunbiotinylated Å6.

Example VI Additional In Vivo Actions of Å6 and Related Peptides

A. Angiogenesis in Chick Chorioaliantoic Membrane (CAM)

Å6 was tested for its effect on bFGF-induced angiogenesis in a 7 day oldchick CAM assay. FIG. 10 shows results that are the average of 10 eggsfor each condition tested. Å6 inhibited angiogenesis in this system. Inaddition to inhibiting major vessel formation, Å6 also inhibitedbranching morphogenesis; this effect is qualitatively evident in thestereomicroscopic images of the CAMs (FIG. 11).

The CAM assay serves as a useful system for measuring the effects of Å6and related peptides or derivatives on events associated with theangiogenic “switch” as these events are observable in near-real time.

B. Tests for Inhibition of Tumor Angiogenesis

Angiogenesis induced by tumor growth and metastasis in vivo is examinedin the models systems described above. Mice injected with 3LL cells aretreated either with peptide or with vehicle and are sacrificed atvarious time points. Angiogenesis is assessed by determining microvesseldensity (MVD) using an antibody specific for microvascular endotheliumor other markers of growing blood vessels, such as PECAM (CD31). Such anantibody is employed in conventional immunohistological methods toimmunostain tissue sections as described by Penfold et al., supra. Alarge number of such antibodies is commercially available, for examplethe JC70 mAb. The MVD are correlated with other measures of tumorbehavior including lymph node status and primary tumor size and rate ofgrowth. In humans as reported by Penfold et al., supra, tumor MVDcorrelates with lymph node metastasis and is independent of tumor size,growth rate or type of histological differentiation. Only MVD showed asignificant association with lymph node metastasis.

The compounds are given i.v., i.p., or by osmotic minipump Typicaldosages are 100-250 mg/kg/day. At various time points, two animals aresacrificed, and the tumor tissue and surrounding tissue is prepared forhistological examination. Results are reported as the averagemicrovessel density of 5 fields each from 5 different sections. Thefollowing seven compounds are tested: Ac-KPSSPPEE-Am (SEQ ID NO:2),Ac-KPTTPPEE-Am (SEQ ID NO: 6) (disubstitution variant at positions 3 and4), Ac-KPSSPPDD-Am (SEQ ID NO: 7) (disubstitution variant at positions 7and 8), Ac-RPSSPPEE-Am (SEQ ID NO: 8) (substitution variant at position1), Ac-PSSPPEE-Am (SEQ ID NO: 4) (deletion variant, position 1 of SEQ IDNO:2 deleted), Ac-KPSSPPE-Am (SEQ ID NO: 5) (deletion variant, position8 of SEQ ID NO:2 deleted), and Ac-KPPSSPPEELK-Am (SEQ ID NO:1).

The following results are obtained. In the rats treated withAc-KPSSPPEE-Am (SEQ ID NO: 2), Ac-KPTTPPEE-Am (SEQ ID NO: 6),Ac-KPSSPPDD-Am (SEQ ID NO: 7) and Ac-RPSSPPEE-Am (SEQ ID NO: 8), thereis a significant reduction in the number of microvessels in the regionof the primary tumor at the subcutaneous inoculation site as compared tocontrols. Peptides Ac-PSSPPEE-Am (SEQ ID NO: 4), Ac-KPSSPPE-Am (SEQ IDNO: 5) and Ac-KPPSSPPEELK-Am (SEQ ID NO: 1) had no significant effect onangiogenesis. Therefore, the four indicated compounds haveanti-angiogenic activity which is responsible at least in part for theireffectiveness as antitumor agents.

C. Growth and Metastasis of Rat Mat B-ITT Breast Cancer

The rat syngeneic breast cancer system (Xing and Rabbani, 1996) employsMat BIIIrat breast cancer cells. When Mat B-III cells (1×10⁶ cells) areinoculated into the mammary fat pad of female Fisher rats, they formlarge tumors which metastasize to regional lymph nodes (LNs) and otherdistal sites within 14-20 days.

Å6 was initially delivered prophylactically (infusion starting on theday of tumor inoculation) using an Alzet mini-pump that delivered 75mg/kg/day. This treatment had substantial anti-tumor activity includinginhibition of tumor growth and LN metastasis. Intraperitoneal deliveryof Å6 (75 mg/kg/day given IP b.i.d.) produced similar results on tumorgrowth (FIG. 12) and metastasis (FIG. 13). Metastasis was quantitated bycounting the number of macroscopic foci without regard for their size.

Å6 was clearly more effective when the tumors were smaller. However,despite the increase in growth rate of the primary challenge tumors, theformation of macroscopic metastatic foci continued to be suppressedduring the course of this treatment schedule. Representative LNs wereexcised from both control and Å6-treated animals, fixed in formalin andembedded in paraffin for histological examination. Tumor cells werefound in LNs from Å6-treated rats, despite the lack of macroscopicmetastases.

Histological analysis of the primary tumor also revealed extensivenecrosis at the tumor periphery. Typically, in the primary tumors, thecentral core is spontaneously necrotic. However, in Å6-treated rats, theperiphery was 50-75% necrosed in all tumors evaluated. The onlyremaining viable tumor cells formed perivascular cuffs around whatappeared to be pre-existing blood vessels. Each of these cuffs wasapproximately 5 cells in width, consistent with Folkman's observationsthat a tumor could expand no farther than 5 cells away from its bloodsupply without initiating the formation of neovessels (Folkman J (1992)Semin Cancer Biol 3:65-7112).

Many of the cells in the Å6-treated groups stained positive in the TUNELassay for apoptosis, indicating that apoptosis of tumor cells wasoccurring. Because TUNEL detects fragmented DNA, other mechanisms ofcell death (including necrosis) might also have contributed.

Factor VIII staining of tumor sections in Å6-treated animals alsorevealed a 40-60% decrease in Factor VIII-positive foci (data notshown).

In a comparative analysis of related peptides, the following resultswill be obtained. In the rats treated with Ac-KPSSPPEE-Am (Å6) (SEQ IDNO: 2), Ac-KPTTPFEE-Am (SEQ ID NO: 6), Ac-KPSSPPDD-Am (SEQ ID NO: 7) andAc-RPSSPPEE-Am (SEQ ID NO: 8), there is a significant reduction in thesize of the primary tumor and in the number of metastases in the spleen,lungs, liver, kidney and lymph nodes (enumerated as discrete foci). Uponhistological and immunohistochemical analysis, it is seen that intreated animals, there is increased necrosis and signs of apoptosis.Large necrotic areas are seen in tumor regions lacking inneovascularization. In contrast, treatment with peptides Ac-PSSPPEE-Am(SEQ ID NO: 4), Ac-KPSSPPE-Am (SEQ ID NO: 5) and Ac-KPPSSPPEELK-Am (SEQID NO: 1) will fail to cause a significant change in tumor size ormetastasis.

D. Treatment of Mat B-III Tumors with Å6+Tamoxifen (“TAM”)

The present inventors evaluated the ability of Å6 to potentiate theactivity of TAM, an anti-estrogen used in the treatment of humanestrogen receptor-positive breast cancer. One promise of anti-angiogenictherapy is the potential for preventing or inhibiting tumorigenesis, aprophylactic outcome, in patient populations at risk for a particulartype of cancer. TAM may be beneficial as a prophylactic treatment forsome patients at risk of developing breast cancer (although this iscontroversial because it TAM could accelerate the formation of certainsubtypes of breast cancer. TAM is part of the accepted treatment regimenin early stage breast cancer. Thus, the combination of ananti-angiogenic agent such as the compounds of this invention, will haveprophylactic and therapeutic effects in on early stage breast cancer.

Estrogen receptor-positive Mat B-III tumors were used to testcombination treatment with Å6 (75 mg/kg/day) and TAM (3 mg/kg/day). Incontrast to previous studies, the Mat B-III tumors (inoculated in themammary fat pad) were staged to 40-50 mm³ prior to the initiation oftreatment. Treatment was continued for 8 days during which time primarytumor growth was measured using calipers (FIG. 14). The combination wasa more potent antitumor therapeutic than TAM or Å6 alone.

E. Treatment of Xenografled Human MDA-MB-231 Tumors with Å6

The results obtained with the Mat B-III model were extended to a humantumor xenograft model of MDA-MB-231 human breast cancer cells. Thesecells were first transfected with green fluorescent protein (GFP) tosimplify the detection and visualization of metastases. The growthcurves of the GFP-transfectedMDA-MB-231-cells-(MDA-MB-23-1 GFP) wereindistinguishable from the parental cells. The in vitro invasiveactivity of both cell lines was also identical indicating that GFPtransfection did not alter cellular behavior,

The tumor cells (5×10⁵) were suspended in 0.1 mL of Matrigel andinjected into the mammary fat pad of female BALB/c nu/nu mice. Treatment(75 mg/kg/day) was initiated when the tumors were palpable (10 mm³,approximately 4 weeks after inoculation of tumor cells) and continuedfor 5 weeks. Tumor volumes were determined twice per week using calipermeasurements. The animals were euthanized at the end of the 5^(th) weekof treatment, necropsied and examined for macroscopic metastases.Sections prepared from LNs, lung, liver, spleen and kidney were analyzedfor microscopic dissemination of tumor cells using fluorescencemicroscopy to detect GFP-positive foci. As shown in FIG. 15, Å6treatment inhibited tumor growth by >80%.

Å6 was ineffective in this model if the tumors were staged to 50-100 mm³prior to initiating treatment. The bi-phasic nature of the growth curveappears to represent the growth rate of the tumor before (slow growth ordormancy) and after (fast growth) the angiogenic switch (which occurs atthe inflection point of the curve). Thus, if Å6 is found to inhibitevents associated with the angiogenic switch, to be effective it shouldbe administered before the switch. Folkman's group (Bergers et al.,Science 284: 808-812) recently demonstrated the stage-specific nature ofangiogenesis inhibitors. Most angiogenesis inhibitors do not causeregression of established tumors when used alone—anti-angiogenic therapyappears to be most efficacious in animal models when it is targeted to aspecific stage of tumor progression. The activity of Å6 is consistentwith this notion.

TUNEL and Factor VIII staining of tumor sections revealed resultssimilar to those observed in the rat studies reported above. Tumors fromÅ6-treated animals demonstrated a significant increase in TUNEL-positivefoci as well as a decrease in Factor VIII-positive hot spots.

Macroscopic metastases were enumerated and their size measured withcalipers (Table I below). Microscopic dissemination of tumor cells wasquantitated by counting GFP-positive foci in representative sectionsfrom lung, liver, kidney and spleen. Disseminated tumor cells were notevident in kidney sections. Because disseminated cells exist in adormant state for many years or, in some cases, never progress, onecannot posit an absolute correlation between the presence ofdisseminated cells and metastasis. Nevertheless, the foci in liver andlungs did appear to be true metastases as the cells were not single focibut seem to have formed larger colonies in the control animals. Sectionsfrom Å6-treated animals appeared to have a greater number of single foci(vs. larger colonies), indicating lack of metastatic progression.

Macrometastases were determined by excising involved LNs and determiningthe tumor volume by caliper measurement. Microscopic tumor foci werequantitated by sectioning and fixing target organs, then visualizingGFP-labeled cells using fluorescence microscopy (at 200× enlargement).The number of disseminated tumor foci represents the average of 5 fieldsper section from 3 different sections per organ.

TABLE I (a) Macroscopic Metastases Lymph Nodes Lungs Group Number Size(mm³) Number Size Control 4.5 ± 1.2 27 ± 3 4.2 ± 1.8 N.D. Å6-treated 1.2± 0.2  7 ± 3 1 ± 0 N.D. (b) Disseminated GFP-positive Tumor Foci LungLiver Spleen Control 4.23 ± 0.18 18.83 ± 0.36 35.67 ± .82  Å6-treated2.09 ± 0.30  3.11 ± 0.14 28.30 ± 2.80

F. Treatment U87 Human Glioblastoma U87 Xenografts

1. Growth of Primary Tumors after Subcutaneous Implantation

Tumors were established by subcutaneous injection of human U87glioblastoma (“GBM”) cells sc into nude mice. U87 tumors were staged to50-100 mm³ prior to initiating treatment. Å6, cisplatin (CDDP) and thecombination of Å6+CDDP were tested (FIG. 16). CDDP was tested at a doseof 3 mg/kg/day given every other day from day 4×6 administrations, whichconverts to approximately 6 mg/m² per administration. This is asubstantially lower dose than that typically given to human patients(20-40 mg/m² for most tumors although doses as high as 200 mg/m² havebeen reported in neuroblastomas; the dose depends on the regimen used)since dose limiting toxicity (as exemplified by weight loss) occurs inmice at doses greater than 6 mg/m². Thus, the full benefit of CDDP+Å6 asa combination therapy may exceed that which was observed here.

As the combination treatment of Å6+CDDP was highly effective ininhibiting tumor growth in this model the present inventors assessed theproliferative (mitotic) index in these tumors. Typically, human GBM isnot characterized by rapid proliferation and only 15% of the tumor cellsare typically proliferating (CLINICAL ONCOLOGY (1995) Abeloff; M. D. etal., eds. Churchill-Livingstone, New York). For this reason,anti-metabolites are not efficacious in treating this type of tumor.Though alkylating agents (such as CDDP and BCNU) have been the mostsuccessful in treating GBM in the clinic (as they induce apoptosis bydamaging tumor cell DNA), they are not selective for rapidly dividingcell populations and are quite toxic.

Anti-angiogenic therapy is expected to produce both anti-proliferativeand pro-apoptotic effects that would “prime” the tumor chemotherapy withthese alkylating agents. The proliferative index in the U87 tumors wasevaluated using Ki-67 staining followed by digitization of the stainingintensity and quantitation. Three sections from each animal wereevaluated for Ki-67 positive staining (FIG. 18). Å6 treatment inhibitedproliferation by 50%, which was not enhanced by combination treatmentwith CDDP, as predicted by the inventors. Å6 did not inhibit U87 cellproliferation directly nor did it potentiate the pro-apoptotic activityof CDDP in vitro. Similar results were obtained against other human GBMcell lines, including those designated 308 and U251.

Dose Response Studies

The effect of different doses of Å6 on U87 tumor growth sc was evaluated(FIG. 19). Tumor growth was almost completely suppressed in animalstreated with the combination of the Å6 (150 mg/kg/day) and CDDP as longas treatment was continued. Tumors grew when treatment was discontinued.Tumor regression (defined as a tumor that was no longer palpable) wasobserved in 1 or 4 mice in this group, a response that was durablethroughout the course of the experiment. This individual mouse is beinganalyzed for the presence of microscopic tumor. Once information with aneven higher dose of Å6 (300 mg/kg/day) been obtained, the results willbe extrapolated to the orthotopic model where the effects of higherdoses of Å6 on angiogenesis and survival will be evaluated.

2. U87 Implanted Orthotopically

U87 tumors were surgically inoculated into the cerebral ventricles ofmice. The animals were allowed to recover for 72 hours at which timetreatment with Å6 (75 mg/kg/day IP bid), CDDP (3 mg/kg/day given everyother day from day 4×6 administrations), or a combination of Å6+CDDP wasinitiated,

Animals were treated for 21 days at which time they were euthanized andtheir brains evaluated for the presence of tumor. Transverse sectionswere stained with hematoxylin and eosin (H&E). The combination of Å6 andCDDP was significantly more effective in inhibiting U87 tumor growththan either agent alone.

Tumor sections were also evaluated for microvessel density usingantibodies specific for mouse-CD31 in immunostaining. Very few CD31⁺foci were evident in animals treated with the combination of Å6+CDDP. Infact, CDDP and Å6 alone both inhibited angiogenesis to some extent.Qualitatively, Å6 appeared not only to inhibit the number of vessels butalso the differentiation of the vessels as fewer branching vessels wereobserved in the Å6-treated tumors.

3. Survival of Mice Implanted Orthotopically with U87

Tumors were established as in the previous section, and the sametherapeutic regimens were employed. Treatment was discontinued at day21, and survival was measured. Control animals appeared moribund aroundday 25 and all of the animals in this group were dead by day 30. Incontrast, the combination treatment group showed a significant increasein survival when compared to the control group or to the groupsreceiving either Å6 or CDDP alone (FIG. 20).

G. Treatment of Murinc Lewis Lung Carcinoma (3LL)

Experimental Metastasis/Lung Colonization

Lewis Lung Carcinoma cells (3LL, 1.5×10⁵ cells per mouse) were injectedi.v. into C57BL/6 mice. Mice were treated with cyclophosphamide (CY: 300mg/kg once on day 4) alone, Å6 alone (treated from day 0-day 19 at 75mg/kg/day) or a combination of CY+Å6. The animals were euthanized on day19 and the lungs harvested for analysis. Macroscopic metastases werecounted and the lungs analyzed histologically. Combination treatmentreduced the total number of lung colonies when compared to control or CYonly groups (FIG. 21).

Histological evaluation of tumors from the combination treatment grouprevealed that the lungs were mostly free of tumor. In lungs having smallamounts of residual tumor, hemorrhagic necrosis was observed in some ofthe foci.

Additional Studies of Metastasis

In addition to Å6, other related peptide compounds described above aretested for efficacy in vivo in the 3LL model (as provided above) as wellas the PC-3 model. PC-3 cells transfected with the gene encoding theenzyme chloramphenicol acetyl-transferase (CAT) are inoculated into micei.v. at doses of 1×10⁶ cells per mouse. These mice are implanted with aminipump, as above, which dispenses 100 mg/kg/day of the peptide orvehicle over a period of 14 or 21 days. At termination of treatment, theanimals are euthanized and the tumor marker probe is assayed in regionallymph nodes, femurs, lungs, and brain.

The following results are obtained in both systems. In mice treated withAc-KPSSPPEE-Am (SEQ ID NO: 2) (Å6), Ac-KPTTPPEE-Am (SEQ ID NO: 6),Ac-KPSSPPDD-Am (SEQ ID NO: 7) and Ac-RPSSPPEE-Am (SEQ ID NO: 8),metastasis is markedly inhibited. These results indicate that thesecompounds interfere with the metastatic process. In contrast, micetreated with peptides Ac-PSSPPEE-Am (SEQ ID NO: 4) Ac-KPSSPPE-Am (SEQ IDNO: 5) and Ac-KPPSSPPEELK-Am (SEQ ID NO: 1) have no reduction inmetastases.

Other uPAR-binding peptides with utility as tumor-targeted imagingagents that have been discovered by the present inventors are thesubject of an issued (U.S. Pat. No. 5,942,492) and co-pending, commonlyassigned U.S. Ser. No. 09/285,783 filed 5 Apr. 1999; U.S. Ser. No.09/181,816 filed 29 Oct. 1999; and provisional application 60/157,012,filed 1 Oct. 1999 (all of which documents are incorporated by referencein their entirety). Some of these agents are useful as radiodiagnosticsfor evaluating tumor size and dissemination. These can be used inmonitoring patients during therapy. Some of these reagents have beendesigned to carry a Tc^(99m) γ-emitting nuclide to the surface of solidtumors and tumor vessels. These same constructs are also useful inlocalizing a Tc⁹⁴ isotope for PET imaging.

Example VII Pharmacological and Pharmacodynamic Evaluation of Å6

1. Pharmacodyamic Markers

Evaluation has begun of several markers that are expected to beassociated with a therapeutic response of tumors to Å6. Moleculesassociated with angiogenesis will be evaluated for their utility aspharmacodynamic markers of Å6 activity. Certain markers bFGF, MMPs) willbe detected in urine

2. Pharmacology

Liquid Chromatography/Mass Spectroscopy (LC/MS) Assay Development

An LC/MS assay developed for the detection of Å6 in plasma has beenvalidated for mouse and monkey plasma. The sensitivity is 10 ng in 0.1mL of plasma. This test can be similarly validated for human plasmaunder GLP conditions to be used for human pharmacology. Protein bindingstudies are presently underway. Because the peptide can be recoveredfrom plasma after simple precipitation of total protein (Å6 is in thesupernatant), the undesired effect caused by extensive binding of thepeptide to other proteins is not expected. Å6 is extremely soluble andcan be formulated in physiological buffers and excipients to highconcentration (>100 mg/mL).

Plasma Stability of Å6

Å6 was stable in plasma at room temperature for 24 hrs (tested at 10μg/mL). Å6 is stable as a lyophilized powder for at least 2 months(degradation is <1% by HPLC) and in PBS or water at 4° C. for at least 2weeks.

Pharmacokinetics in Mice

Pharmacokinetic analysis was carried out by Gilbert Lam, MicroConstants.The plasma concentration profile of Å6 is depicted in FIG. 22. Thepharmacokinetics of Å6 is characterized by a mono-exponential declinefollowing a single bolus dose. The terminal half-life is 0.2 hour. Thesystemic clearance “CL” is 2.0 L/h/kg, which is moderate when comparedto the liver blood flow of the mouse. Å6 has a small volume ofdistribution at steady-state, (V_(ss)=0.4064 L/kg). Low V_(ss) resultsin high plasma concentrations of the peptide.

Pharmacokinetics in Monkeys

The plasma concentration profile of Å6 is depicted in FIG. 23. Å6pharmacokinetics is characterized by a mono-exponential declinefollowing a single bolus dose. The terminal half-life is 0.4 hour. Thesystemic clearance, CL, is 0.042 L/h/kg which is low when compared toeither the kidney blood flow or the liver blood flow of the monkey. Å6has a small volume of distribution at steady-state, V_(ss)=0.0252 L/kg.Low V_(ss) results in high plasma concentrations of the peptide.

Mouse Plasma Therapeutic Levels

The therapeutic plasma levels in mice receiving Å6 are being evaluated.Plasma concentration of Å6 will be measured in blood samples obtainedfrom the dose-response studies in the glioblastoma model (above). Thisinformation is combined with allometric scaling data (see below) topredict therapeutic doses in man. This information will then be combinedwith toxicology data to establish starting doses for a Phase I trial.

Allometric Scaling

Å6 is a particularly good candidate for allometric scaling analysis. Thedistribution of this compound is simple (i.e. after iv administration,the compound is not absorbed by, or distributed in, tissue but rather isrestricted to the plasma) and is proportional to the plasma volume andtherefore body weight. This makes the prediction of clearance in manfairly straightforward. Based on allometric scaling, it is expected thatthe CL in man is about 0.0061 L/h/kg (0.43 L/h for a 70 kg person) (FIG.24). Extrapolations can also be made for other parameters such ast_(1/2).

3. Toxicology

Acute toxicity was evaluated at three different doses of Å6 (1500 mg/kg,500 mg/kg and 250 mg/kg). Mice (n=6) were infused (i.v,) with a bolus ofÅ6 over 5-10 minutes and observed for 3 days. No evidence for overttoxicity was observed, and all the animals survived and apparentlytolerated the Å6 well.

DOCUMENTS CITED

A number of documents are cited only in the text above (in full).Others, cited in abbreviated form in the text, are cited in full below.

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The references cited above are all incorporated by reference herein,whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

1. A method for inhibiting cell migration, invasion, migration-inducedcell proliferation or angiogenesis in a subject having a disease orcondition associated with undesired cell migration, invasion,migration-induced proliferation, or angiogenesis, comprisingadministering to said subject an effective amount of a pharmaceuticalcomposition comprising (a) a peptide consisting of the capped sequenceAc-SEQ ID NO:2-Am (b) a pharmaceutically acceptable carrier orexcipient; wherein said disease or condition is atherosclerosis.
 2. Themethod of claim 1, wherein said peptide is labeled.
 3. The method ofclaim 2, wherein said label is selected from a radioactive, fluorogenic,chromogenic, or other chemical label.
 4. The method of claim 1, whereinsaid peptide is conjugated to a therapeutic agent.